Legend to Supplementary Figures
Supplementary Figure 1
SH2B1β co-expression reverses the blockade of RET-dependent transactivation by
tyrosine kinase inhibitors. HeLa cells were transfected with FLAG-STAT3 and with the
pm67luc reporter plasmid in presence or absence of SH2B1β expression vector, treated
for 18-20 hrs with the indicated tyrosine kinase inhibitors in order to impair RET
activation of STAT3 effector, and luciferase activity was assayed. STAT3-associated
reporter activity, elicited by RET, was significantly decreased by TK inhibitors, while
SH2B1β co-transfection was effective in quelling their effect on pm67luc transactivation.
The average percent of residual activity following inhibition of RET is reported on the
corresponding bar. Data (n = 3) are expressed as means ± S.E. S3, STAT3. PTC,
RET/PTC2 iso9.
Supplementary Figure 2
RET steady-state level appears up-regulated by wild-type SH2B1β. Cells co-expressing
distinct combination of RET/PTC2 and the adaptor, were subjected to time-course
cycloeximide (CHX, 10 μg/mL) treatment, and protein levels evaluated by
immunoblotting. Co-transfection of SH2B1β, but not its inactive SH2B1βRE mutant,
increases RET/PTC2 for the 9 hrs of time-course experiments. Exogenous SH2B1β-wt
had no valuable effect on RET half-life over its RE counterpart.
Supplementary Figure 3
SH2B1β increases the RET-induced phosphoproteoma. Lysates from 293T cells
transfected with empty vector (mock), RET/PTC2 or RET/PTC2 plus SH2B1β were
affinity-purified with immobilized αpTyr. Phosphorylated bound proteins were resolved
on SDS-PAGE (4-12%) and detected by silver-staining, before isolated proteins were
1
identified by peptide mass fingerprinting analysis. RET/PTC2 and SH2B1β bands are
indicated. The list summarizes the proteins identified in the different transfected cells.
Band number refers to specific band position on the gels; each band can contain more
than one protein.
Supplementary Figure 4
RET-driven differentiation of PC12 pheocromocytoma cells in the presence of SH2B1β.
(a) Phase-contrast micrographs of transfected PC12 cells (magnification x 10). The cells
were transfected as described in Materials and Methods and treated with 10 ng/ml of
GDNF for 2 days, when indicated. (b) PC12 cells, seeded at 200,000 cell/well (12-wells
plates), were transfected with pvgf8luc reporter vector in combination with the indicated
constructs. The day after cells were treated or not with GDNF and 48 hours later assayed
for luciferase activity. The data represent the mean values ± S.E. of triplicate samples.
Supplementary Figure 5
Activation of MAPK/ERK pathway in PC12 pheocromocytoma by RET/SH2B1β
complex. Lysates from cells were transiently transfected with RET/PTC2 mutant and
GFP-SH2B1β were immunoblotted and the extent of ERK1/2 phosphorylation was
assessed by αpERK. Phosphorylation of ERK1/2 proteins by SH2B1β and RET is
significant in cells transfected with RET/PTC2wt, RET/PTC2-Y505F and -Y586F. A
slight phosphorylation is achieved by RET/PTC2Y429F, and no phosphorylation by
RET/PTC2Y429F/Y505F in presence of the adaptor.
Supplementary Figure 6
Biochemical characterization of SH2-Bβ 24-88 and Y439F/Y494F mutants. Lysates
from 293T cells transfected with RET/PTC2 and SH2B1 constructs were
2
immunoprecipitated with anti-RET or anti-GFP antibody, and samples immunoblotted
with
anti-RET,
anti-GFP
or
anti-phosphotyrosine
(αpTyr)
antibodies.
IP,
immunoprecipitation. WB, Western blotting; WCL, whole cell lysate; RE, R555E
mutant.
3