Oncogene (1998) 16, 211 ± 216
 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00
Regulation of c-myc expression by Ras/Raf signalling
Eugen Kerkho€1,*, Roland Houben1,*, Silke LoȂer1, Jakob Troppmair1, Jong-Eun Lee2 and
Ulf R. Rapp1
1
2
Institut fuÈr medizinische Strahlenkunde und Zellforschung, University of WuÈrzburg, Versbacher Str. 5, 97078 WuÈrzburg, Germany;
Eunpyung-Gu, Nokbeon-Dong 35-5, Seoul, Korea 122-020
The c-myc gene is induced upon growth factor
stimulation of arrested cells. The interaction of a
mitogen with a transmembrane receptor triggers a
variety of parallel signal transduction cascades. In order
to analyse the role of the Ras/Raf cascade in the
regulation of c-myc expression we have established
®broblast cell lines harboring conditional systems
activating or inhibiting this pathway. Fusion of the cRaf-1 kinase domain with the hormone binding domain
of the estrogen receptor (c-Raf-1-BxB-ERTM) provides a
4-hydroxytamoxifen regulated form of the oncogenic cRaf-1 kinase. We have generated NIH3T3 cells stably
expressing the chimeric Raf protein (N-BxB-ERTM). 4hydroxytamoxifen mediated activation of the fusion
protein in serum starved N-BxB-ERTM induces the
expression of the c-myc gene within 2 ± 6 h. Deletion of
the c-Raf-1 kinase domain generates a mutant c-Raf-1
protein (c-Raf-1-C4B), which can directly interact with
the e€ector domain of the Ras protein and thereby block
Ras mediated signalling. We have established a NIH3T3
based cell line expressing the c-Raf-1-C4B protein under
the control of a tetracycline responsive promoter (NC4B-tet). Serum starved cells expressing the c-Raf-1C4B protein exhibit a signi®cantly reduced induction of
c-myc expression following serum stimulation compared
to the same cells not expressing the Ras inhibitor. The
induction of c-myc mRNA following the activation of the
isolated Raf/Mek/Erk cascade in addition to the partial
inhibition of serum mediated induction of c-myc
expression in the presence of the Ras inactivating cRaf-1-C4B mutant strongly indicates an involvement of
the Ras/Raf pathway in the regulation of c-myc
expression.
Keywords: raf; signal transduction; myc
Introduction
Activation of a growth factor receptor stimulates a
variety of parallel signal transduction cascades. These
cascades shuttle the signal into the nucleus, where the
expression of genes necessary for cell cycle progression is initiated. One of those genes that become
activated early after growth factor stimulation of
arrested cells is the c-myc gene (MuÈller et al., 1984).
The c-myc gene encodes a sequence speci®c DNAbinding protein which is involved in control of gene
transcription (Amati and Land, 1994). The expression
Correspondence: UR Rapp
*These authors contributed equally to the work
Received 18 June 1997; revised 27 August 1997; accepted 27 August
1997
of the c-Myc protein correlates with the proliferative
status of cells and its function has been shown to be
necessary for cell proliferation (Waters et al., 1991;
Heikkila et al., 1987). The overexpression of the cMyc protein can contribute to the oncogenic
transformation of cells (Cooper, 1995). The Ras
GTPase plays a central role in growth factor induced
signal transduction events. Mutations generating a
constitutively active form of the Ras protein are
highly oncogenic and are frequently found in human
cancer. Inhibition of Ras function blocks cell
proliferation (Dobrowolski et al., 1994). Oncogenic
Ras and Myc cooperate in the transformation of
primary rat embryo ®broblasts (Land et al., 1983),
leading to the opinion that c-Myc and Ras are on
separate pathways. Recently the regulation of c-myc
expression has been discussed controversially. Data
showing that a block in S phase induction by
dominant negative Src or Ras mutants can be
overcome by Myc overexpression only in the case of
the dominant negative Src argues that a Src and not
Ras mediated signaltransduction pathway is responsible for the c-myc induction (Barone and Courtneidge, 1995). However there is also evidence that the
c-myc gene is regulated by Ras mediated signalling.
De®ciencies in the Ras pathway caused by overexpression of the carboxy-terminal catalytic domain of
the GTPase-activating protein (GAP) has been shown
to be rescued by c-myc overexpression (Bortner et al.,
1992). Furthermore the human and mouse c-myc
promoters contain binding sites for the Ets transcription factors (Roussel et al., 1994). Ets-1 can
transactivate reporter genes driven by the human
and mouse c-myc promoters (Roussel et al., 1994).
The Ets-1 and Ets-2 transcription factors can be
activated by Ras/Raf/Mek/Erk signalling (McCharty
et al., 1997; Wasylyk et al., 1997; Yang et al., 1996),
indicating a role for Ras/Raf signalling in the
regulation of the c-myc gene. By employing conditional cell lines harboring an inducible Ras inhibitory
protein and an inducible constitutive active form of
the c-Raf-1 protein we have addressed the question if
the Ras/Raf signal transduction pathway is involved
in the regulation of the c-myc gene expression.
Results
The c-Raf-1 protein has a N-terminal regulatory
domain and a C-terminal kinase domain (Figure 1).
The regulatory domain interacts directly with the Ras
e€ector domain (Daum et al., 1994). By this interaction
the c-Raf-1 protein becomes translocated to the cellular
membrane (Leevers et al., 1994). This step has been
shown to be essential in the activation of the c-Raf-1
c-myc regulation by Ras/Raf signalling
E Kerkhoff et al
212
NIH3T3
NIH3T3 / BJ4-Myc-ERTM
NIH3T3 / BJ4-BxB-ERTM
a
N-BxB-ERTM
0 4 8 12 24
OHT induction (h)
c-Raf-1-BxB-ERTM
b
N-C4B-tet
TM
Figure 1 Structure of the c-Raf-1-BxB-ER
and c-Raf-1-C4B
proteins. The c-Raf-1 kinase consists of a regulatory N-terminal
domain which interacts directly with the Ras protein and a
catalytic C-terminal domain. Deletion of the regulatory domain
generates a constitutive active kinase (c-Raf-1-BxB). Fusion of
this deletion mutant to the hormone binding domain provides a 4hydroxytamoxifen (OHT) regulated chimeric kinase (c-Raf-1BxB-ERTM) (Kerkho€ and Rapp, 1997). The c-Raf-1-C4B protein
is a B-Raf tagged (human B-Raf amino acids 750 ± 765) version of
the N-terminal half of the c-Raf-1 protein. c-Raf-1-C4B can
interact with the Ras protein and by this block Ras function
kinase by growth factors. Deletion of the regulatory
domain generates a constitutive active kinase (Stanton
et al., 1989; Heidecker et al., 1990). Fusion of the cRaf-1 kinase domain to the hormone binding domain
of the estrogen receptor provides a hormone regulated
form of the c-Raf-1 kinase (Figure 1) (Samuels et al.,
1993; Kerkho€ and Rapp, 1997). We have established
a NIH3T3 based cell line stably expressing a chimeric
c-Raf-1-estrogen receptor protein (c-Raf-1-BxB-ERTM,
N-BxB-ERTM) (Figure 2a) (Kerkho€ and Rapp, 1997).
Addition of the estrogen antagonist 4-hydroxytamoxifen (OHT) to proliferating or arrested con¯uent NBxB-ERTM cells induces a strong and sustained
activation of the Erk kinases within 1 h (Kerkho€
and Rapp, 1997). Under low serum conditions the
activation of the Erk kinases is less pronounced
(Samuels and McMahon, 1994; Kerkho€ and Rapp,
1997). In addition to the early activation of the c-Raf1-BxB-ERTM kinase activity, the presence of OHT leads
also to an increase in the protein concentration of the
fusion protein within 12 ± 24 h (Figure 2a). Constitutive
activation of the oncogenic c-Raf-1 protein in N-BxBERTM cells has been shown to be sucient to drive cells
arrested by con¯uency or serum starvation back into
the cell cycle (Kerkho€ and Rapp, 1997). In agreement
with the di€erences in Erk activation, the kinetics of
the cell cycle re-entry are delayed in the case of the
serum starved cells.
In order to analyse if the activation of the Ras/Raf
cascade can induce the expression of the c-myc gene in
the absence of the activation of other signal
transduction cascades, we have activated the Raf
pathway in N-BxB-ERTM cells arrested by serum
starvation or cell density. Analyses of mRNA
expression by Northern hybridisation revealed a weak
induction of c-myc expression 2 h following OHT
stimulation (Figure 3). Between 2 and 6 h a strong
increase of c-myc mRNA was detected (Figure 3). The
0
24
48
tet deprivation (h)
c-Raf-1-C4B (31 kD)
c
Figure 2 Expression of the c-Raf-1-BxB-ERTM and c-Raf-1-C4B
proteins in N-BxB-ERTM or N-C4B-tet cells. (a) Total protein
extract of NIH3T3 cells, NIH3T3 cells transiently transfected with
the BJ4-Myc-ERTM or BJ4-BxB-ERTM expression vectors and
con¯uent N-BxB-ERTM cells (Kerkho€ and Rapp, 1997) induced
for 0, 4, 8, 12, and 24 h with OHT were separated by SDS
polyacrylamide gelelectrophoreses and the expression of the cRaf-1-BxB-ERTM protein (80 kDa) was analysed by immunoblotting using polyclonal antibodies of the c-Raf-1-30K rabbit serum.
Equal protein loading was veri®ed by ponceau S staining. (b)
Total protein extracts of N-C4B-tet cells in the presence of
tetracycline and in the absence of tetracycline for 24 and 48 h
were separated by SDS polyacrylamide gelelectrophoreses and the
expression of the c-Raf-1-C4B protein (31 kDa) was analysed by
immunoblotting using polyclonal antibodies of the B-Raf rabbit
serum. Equal protein loading was veri®ed by ponceau S staining.
(c) N-C4B-tet cells were cultured in the presence and absence of
tetracycline for the period of 4 days. Cell numbers were obtained
by counting trypsinized cells with the help of a hematocytometer
levels of induction of c-myc mRNA are lower under
low serum conditions compared to OHT stimulated
con¯uent cells and by this re¯ecting the di€erences we
see in the activation of the Erk kinases and the
initiation of the cell cycle reentry. Addition of serum to
N-BxB-ERTM cells arrested by serum starvation results
in a strong induction of c-myc expression within 1 h of
c-myc regulation by Ras/Raf signalling
E Kerkhoff et al
low / ser 1h
low
pro
213
N-C4B-tet
N-BxB-ERTM
low serum
0 2 6 12
confluent
0 2 6 12
tet
–
+
OHT induction (h)
— c-raf-1-C4B
— c-myc
— c-myc
— c-fos
— c-fos
— 28s
— 28s
Figure 3 Induction of c-myc expression by activation of the cRaf-1-BxB-ERTM protein in arrested N-BxB-ERTM cells. N-BxBERTM cells were arrested in the absence of OHT by serum
deprivation (48 h, 0.05% serum) or con¯uency (24 h after
reaching con¯uency). Total RNA was isolated from proliferating
cells (pro), serum starved cells (low), serum starved cells induced
for 1 h with 10% fetal calf serum (low/ser 1h), serum starved cells
induced with OHT for 0, 2, 6 and 12 h and con¯uent cells
induced with OHT for 0, 2, 6, and 12 h. The RNAs were
separated by agarose gelelectrophoreses and blotted onto a
Duralon membrane. The blotted RNA was hybridized with
a-32P-dATP labelled mouse c-myc or mouse c-fos cDNA probes.
The bands were visualized by autoradiography. For quanti®cation
of the amount of total RNA, the ethidium bromide-stained bands
of the 28s rRNA are shown
Figure 4 Partial repression of c-myc and c-fos expression after
serum stimulation of serum starved N-C4B-tet cells in the
presence of the Ras inhibiting c-Raf-1-C4B protein. N-C4B-tet
cells were cultured under high serum conditions (10%) for 24 h in
the presence and absence of tetracycline. The cells were washed
free of serum and incubated for 6 h under low serum conditions
(0.05%) in the presence and absence of tetracycline. One hour
following the induction of the cells with 1% fetal calf serum, total
RNA was isolated and analysed by Northern hybridisation using
a-32P-dATP labelled human c-raf-1-C4B, mouse c-myc or mouse
c-fos cDNA probes. The bands were visualized by autoradiography. For quanti®cation of the amount of total RNA, the
ethidium bromide-stained bands of the 28s rRNA are shown
stimulation (Figure 3). The c-fos gene is expressed
transiently following growth factor stimulation of
arrested cells (Figure 3). In arrested and proliferating
cells the expression is nearly undetectable (Figure 3).
Although the c-fos promoter has been shown to be
induced by Ras/Raf signalling in transient promoter
assays (Jamal and Zi€, 1990), cells transformed by
oncogenic Raf do not express steady state levels of cfos (Siegfried and Zi€, 1990; Rapp et al., 1994).
Consistent with the observation that the expression of
c-fos is transient in Raf transformed cells we do not see
a signi®cant increase of c-fos mRNA between 2 and
12 h of OHT induction in arrested N-BxB-ERTM cells
(Figure 3).
The isolated regulatory domain of the c-Raf-1
protein (c-Raf-1-C4) (Figure 1) has been shown to
inhibit Ras function by interacting with the Ras
e€ector domain (Bruder et al., 1992; Daum et al.,
1994). We have generated NIH3T3 cells expressing a
tagged version of the c-Raf-1-C4 protein (c-Raf-1-C4B)
under the control of a tetracycline responsive promoter
(N-C4B-tet). In the presence of tetracycline the
expression is repressed (Figure 2b). Removal of the
tetracycline leads to an accumulation of the c-Raf-1C4B protein and inhibits serum mediated proliferation
of these cells (Figure 2b,c). N-C4B-tet cells were
cultured in the presence and absence of tetracycline
and subsequently serum starved for a period of 6 h.
During this period the levels of c-myc mRNA declined
to nearly undetectable levels (data not shown).
Northern hybridisation of RNA samples isolated 1 h
following serum stimulation revealed, that in cells
expressing the c-Raf-1-C4B protein the induction of the
c-myc gene is markedly reduced compared to cells
where the expression of the Ras inhibitor is suppressed
by tetracycline (Figure 4). These results strongly
indicate that Ras function is essential for serum
mediated c-myc expression in NIH3T3 cells. Serum
induced expression of the c-fos gene is also signi®cantly
reduced in the presence of the Ras inhibitor, underlining the role of the Ras signal in the regulation of this
immediate early gene.
A number of groups have reported a cooperation of
the oncogenic Ras or Raf proteins with the c-Myc
protein in cell transformation (Cooper, 1995; Morse
III and Rapp, 1988). While Ras or Raf are
oncogenically activated by mutational constitutive
activation, the c-Myc protein exerts its oncogenic
character by overexpression of the wild type protein.
Our experiments indicate that the Ras/Raf pathway is
involved in the positive regulation of the c-myc gene.
We therefore were interested if oncogenic forms of the
Ras or Raf proteins can oncogenically activate the cmyc gene. We therefore have analysed if activation of
oncogenic Raf in proliferating N-BxB-ERTM cells (high
serum) leads to an overexpression of the c-myc gene
and if the overexpression of an exogenous c-myc gene
causes a more severe transformed phenotype of OHT
stimulated N-BxB-ERTM cells. Induction of the
oncogenic c-Raf-1-BxB-ERTM protein by OHT in NBxB-ERTM cells causes a morphological transformation
of these cells (Figure 5a). In the absence of OHT the
cells exhibit a ¯at non refractile morphology. Addition
of OHT alters the cell shape in that the cells become
spindle shaped and refractile. We have generated NBxB-ERTM cells stably expressing high levels of
exogenous c-Myc protein (N-BxB-ERTM-Myc). The
expression of the exogenous c-myc mRNA and cMyc protein has been veri®ed by Northern hybridisation (Figure 5b) and Western hybridisation (data not
shown). The exogenous c-myc gene in N-BxB-ERTMMyc cells is induced upon OHT addition (Figure 5b).
This is presumably due to the stimulation of the viral
promoter driving the exogenous c-myc gene by the
oncogenic activation of the c-Raf-1-BxB-ERTM protein.
The cells expressing ectopic c-Myc exhibit an
untransformed morphology in the absence of OHT
(Figure 5a). In the presence of OHT however N-BxB-
c-myc regulation by Ras/Raf signalling
E Kerkhoff et al
214
a
N-BXB-ERTM
(-OHT)
N-BXB-ERTM-Myc
(-OHT)
N-BXB-ERTM
(+OHT)
N-BXB-ERTM-Myc
(+OHT)
ERTM-Myc cells have a much more severe transformed
morphology than N-BxB-ERTM cells (Figure 5a). The
overexpression of the c-Myc protein causes the cells to
round up so that they are no longer spindle shaped
and hardly adhering to the tissue culture plate. These
results indicate, that although oncogenic activation of
the c-Raf-1 protein in arrested cells leads to an
induction of c-myc expression, it is not capable to
oncogenically activate the cellular c-myc gene under
high serum conditions, explaining the Raf/Myc
cooperation in cell transformation. This is supported
by the data we obtained from analyses of c-myc
expression in subcon¯uent N-BxB-ERTM cells proliferating under high serum conditions in the presence and
absence of OHT. Oncogenic activation of the c-Raf-1BxB-ERTM protein does not lead to an increase of
endogenous c-myc expression under those conditions
(Figure 5b, N-BxB-ERTM+,- OHT).
Discussion
N-BXB-ERTM-Myc
N-BxB-ERTM
N-BXB-ERTM-Myc
OHT
N-BxB-ERTM
b
+
+
–
–
c-myc (exo)
c-myc (endo)
28s
Figure 5 Overexpression of an exogenous c-myc gene enhances
the transformed phenotype of N-BxB-ERTM cells. (a) N-BxBERTM cells were infected with replication incompetent retroviruses expressing the c-myc gene and a neomycin resistance gene.
N-BxB-ERTM-Myc cells were obtained by G418 selection of the
infected cells. The expression of the exogenous c-myc gene was
veri®ed by Northern hybridization (see below). In the absence of
OHT N-BxB-ERTM and N-BxB-ERTM-Myc exhibit an untransformed non refractile morphology. Induction of the c-Raf-1-BxBERTM kinase by OHT causes morphological transformation of
both cell lines. N-BxB-ERTM cells exhibit a spindle shaped
refractile morphology. The overexpression of the exogenous c-myc
gene enhances the transformed phenotype in that the cells round
up and hardly adhere to the tissue culture plate. (b) The
expression of exogenous and endogenous c-myc genes in NBxB-ERTM and N-BxB-ERTM-Myc cells was analysed by
Northern hybridization. Total RNA was isolated from subcon¯uent N-BxB-ERTM and N-BxB-ERTM-Myc cells, cultured under
high serum conditions (10%) in the presence (+) and absence
(7) of OHT. The RNAs were separated by agarose gelelectrophoreses and blotted onto a Duralon membrane. The blotted
RNA was hybridized with an a-32P-dATP labelled mouse c-myc
cDNA probe. The bands were visualized by autoradiography. For
quanti®cation of the amount of total RNA, the ethidium
bromide-stained bands of the 28s rRNA are shown
The regulation of the c-myc gene has been discussed
controversely in the past. Data supporting a role of the
Ras/Raf signaltransduction cascade in this process
have been published (Bortner et al., 1992; Roussel et
al., 1994; McCharty et al., 1997; Wasylyk et al., 1997)
as well as data showing that a Src and not a Ras
dependent pathway is essential for growth factor
mediated c-myc induction (Barone and Courtneidge,
1995). In order to analyse the role of the Ras/Raf
signaltransduction cascade in the regulation of the cmyc gene we have established stable conditional cell
lines where we can speci®cally activate the Raf
pathway or inhibit Ras function by external stimuli.
The Raf/Mek/Erk signaltransduction cascade is one of
several parallel cascades activated by Ras signalling
(Daum et al., 1994; Avruch et al., 1994; Rodriguez
Viciana et al., 1996; Symons 1996). Induction of the cRaf-BxB-ERTM protein in N-BxB-ERTM cells by OHT
leads to a rapid and sustained induction of the Erk
kinases. With a delay of 2 ± 6 h this signal leads to an
induction of c-myc expression. The oncogenic c-Raf-1
kinase is not able to directly activate other parallel
signaltransduction cascades like the Mekk/Sek/Jnk
cascade during this time period (Minden et al., 1994).
Our results therefore show that the isolated Raf/Mek/
Erk signaltransduction cascade is sucient to induce
the expression of the c-myc gene. These results strongly
support a role of Raf in the induction of c-myc
expression. The mechanism how Raf signalling
activates c-myc expression remains to be established.
The induction kinetics of 2 ± 6 h do not exclude a direct
activation of the c-myc promoter by Erk mediated
activation of the Ets transcription factors. However a
more indirect multistep process could also be possible.
The isolated N-terminal regulatory domain of the cRaf-1 protein binds to the Ras protein and thereby
inhibits its function. Expression of this isolated domain
provided further evidence for the involvement of Ras/
Raf signalling in the regulation of c-myc expression as
its induction in NIH3T3 cells partially inhibited serum
induced c-myc expression in arrested N-C4B-tet cells.
Analysis of the expression c-myc gene in Raf
transformed N-BxB-ERTM cells revealed that the
oncogenic activation of the c-Raf-1 protein is not
c-myc regulation by Ras/Raf signalling
E Kerkhoff et al
able to induce overexpression of c-myc. This explains
the cooperation of the myc and raf oncogenes in
cellular transformation, which originally lead us to the
hypothesis that Raf and Myc are on di€erent pathways. Taken together our results provide strong
evidence that the Ras and Raf signaltransducers are
involved in positive regulation of the c-myc promoter.
The downstream regulatory elements however remain
to be analysed in order to get a more complete
understanding of the regulation of the c-myc gene.
vector (Gossen and Bujard, 1992). Following puromycin
selection (6 mg/ml) in the presence of tetracycline (1 mg/ml),
clones were tested for tetracycline regulated expression of
the c-Raf-1-C4B protein by Western hybridization. N-BxBERTM based cells were routinely cultured in DMEM
medium (Gibco) supplemented with 10% fetalcalf serum
(PAA), 100 units/ml penicillin (Gibco), 100 mg/ml streptomycin (Gibco) and 200 nM OHT. N-C4B-tet cells cultured
in DMEM supplemented with 10% fetal calf serum (PAA),
100 units/ml penicillin (Gibco), 100 mg/ml streptomycin
(Gibco) and 1 mg/ml tetracycline.
Northern blotting
Materials and methods
Cell lines
N-BxB-ERTM cells were obtained by liposome mediated
transfection (Lipofectamine, Gibco) of NIH3T3 cells with
the BJ4-BxB-ERTM plasmid (Kerkho€ and Rapp, 1997).
After transfection the cells were cultured in the presence of
4-hydroxytamoxifen (OHT). N-BxB-ERTM cells were then
isolated from a focus of transformed cells. N-BxB-ERTMMyc cells were obtained by infection of N-BxB-ER TM cells
with replication incompetent retroviruses expressing the cmyc gene and the neomycin resistance gene as a selection
marker. Infected cells were selected in the presence of
500 mg/ml G418. Retroviruses were obtained from the
supernatant of GP+E packaging cells (Markowitz et al.,
1988) transfected by lipofection (Lipofectamine, Gibco)
with the pSRMSVTKneo-c-myc vector (Sawyers et al.,
1992). For enrichment of transfected virus producing cells,
the cells were cultured for two weeks in the presence of
500 mg/ml G418. For the generation of N-C4B-tet cells,
NIH3T3 cells were ®rst transfected by a standard calcium
phosphate precipitation method with the pUHD-15-1
plasmid (Gossen and Bujard, 1992) expressing a tet
repressor-VP16 fusion protein and the pSV-neo plasmid
mediating G418 resistance. For the selection the cells were
cultured for two weeks in the presence of 500 mg/ml G418.
Selected clones were further transfected by lipofection
(Lipofectamine, Gibco) with the pUHG-10-3-C4B vector
and the pBabe-puro vector (Morgenstern and Land, 1990).
The pUHG-10-3-C4B vector was constructed by inserting a
XhoI ± XbaI fragment from the RSV-C4B vector (Bruder et
al., 1992) into the SacII ± XbaI sites of the pUHG-10-3
RNA analyses were performed as described earlier
(Kerkho€ and Rapp, 1997). The following cDNA probes
have been used: c-myc, 1.0 kb XbaI ± SacI fragment of
pSV2-myc (Kelekar and Cole, 1986); c-fos, 1.4 kb EcoRI
fragment of c-fos-Deg-14 (a kind gift from Rodrigo
Bravo), c-Raf-1-C4B, 1 kb XhoI ± XbaI fragment of RSVC4B (Rapp lab). For reuse of the membrane the hybridized
labelled DNA probes were stripped o€ by pouring a
boiling solution containing 15 mM sodium chloride,
1.5 mM sodium citrate and 0.1% SDS over the membrane. The membrane was incubated in the solution for
15 min on a shaker. The procedure was repeated twice.
Immunoblotting
Total cellular protein extracts have been obtained by lysing
the cells in protein sample bu€er (60 mM Tris-HCl pH 6.8,
10% (w/v) Glycerin, 3% (w/v) SDS, 5% (w/v) 2mercaptoethanol, 0.005% (w/v) bromphenolblue). Western blot experiments were performed as described earlier
(Kerkho€ and Rapp, 1997). For immunodetection the
following antibodies were used: c-Raf-1, 30K, rabbit
polyclonal, 1 : 750 dilution, Rapp lab; B-Raf, rabbit
polyclonal, 1 : 1000, Rapp lab.
Acknowledgements
We thank Barbara Bauer for excellent technical assistance.
We further thank Silvia PfraÈnger for excellent photographic reproduction. This work was supported by a grant
of the Wilhelm Sander-Stiftung and the Deutsche Forschungsgemeinschaft (SFB 465 and SFB 172).
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