Protein extraction and western blotting. Cells were lysed in either RIPA or Gold lysis buffer
(GLB) containing the Complete EDTA-free protease and PhosSTOP inhibitor cocktails (Roche
Diagnostics GmbH, Mannheim, Germany), as described previously.(8) Protein concentration was
determined by the Dc Protein Assay (Bio-Rad, Hercules, CA, USA) and 80 g of lysate were
separated by SDS-PAGE. Gels were transferred onto nitrocellulose membranes, blocked in 3%
bovine serum albumin (BSA) in 1X PBS containing 0.05% Tween-20 (PBST) and then incubated in
primary antibody in 3% BSA in PBST [from Cell Signaling Laboratories, Danvers, MA, USA: -pT451 PKR (1:1000); Santa Cruz Biotechnologies (SCB), La Jolla, CA, USA: -PKR (D-20;
1:1000),-PKR (K-17; 1:1000), -eIF2 (FL-315; 1:1000) and -lamin B (1:1000); Abcam,
Cambridge, UK: -N-terminal PKR (ab58301; 1:500) and -C-terminal PKR (ab28943; 1:500) or
Sigma-Aldrich, St. Louis, MO, USA: -p-T451 PKR (1:1000) and --tubulin (1:5000)].
Membranes were washed in 1X PBST and incubated for 1 h in the appropriate secondary antibody
(SCB) diluted 1:10000 in 3% BSA in PBST or 1:2000 in non-fat dry milk in PBST (p-T451 PKR).
The blots were washed three times with 1X PBST, and visualized using SuperSignal West Pico
Reagent (Pierce, Rockford, IL, USA). Band intensity was determined by densitometry using the
public domain software Image J.
Cellular Fractionation and Isolation of Nuclei. Cells (5-10 x 106) were collected by
centrifugation, washed twice in ice cold 1X PBS, pH 7.5 and resuspended in 5X the packed cell
volume in ice cold hypotonic swelling buffer (10 mM HEPES, pH 7.5, 1.5 mM MgCl2, 10 mM
KCl). After 10 min, swollen cells were re-centrifuged, and the pellet was resuspended at 2X the
pellet volume in hypotonic lysis buffer. The suspension was passed 25-30 times through a 27-gauge
needle with syringe. Fractions were isolated with a series of centrifugations at 40C: (centrifugation
1) 400 x g for 5 min to remove intact cells; (centrifugation 2) 2,000 x g for 10 min (nuclear
fraction); (centrifugation 3) 10,000 x g for 15 min (heavy membrane). Supernatant following
centrifugation 3 was saved as the cytoplasmic fraction. Nuclear and heavy membrane pellets were
lysed in GLB as described above. Alternatively, for purification of nuclei, the initial cell pellets
were resuspended in 5X the packed cell volume of hypotonic buffer (10 mM Tris-HCl, pH 7.5, 5
mM MgCl2, 10 mM NaCl) and placed on ice for 5 min. Triton X-100 (10% stock) was added to a
final concentration of 0.3%. The suspension was passed twice through a 22-gauge needle with
syringe and centrifuged at 800 x g for 10 min at 40C to pellet nuclei. Isolated nuclei were lysed in
either RIPA or GLB as described above. The supernatant was centrifuged at 10,000 x g for 10 min
at 40C to removed debris, protease and phosphatase inhibitor cocktails (Roche) as well as glycerol
(15%) were added, and the cleared supernatant was saved as the cytosolic fraction. The protein
concentration in each fraction was determined as stated above.
Liquid Phase Ion Exchange Chromatography. Whole cells or isolated nuclei were solubilized in
2.5 ml of lysis buffer: 50 mM Tris base, 7 M urea, 2 M thiourea, 4% (w/v) n-octyl β-dglucopyranoside, 10 mM TCEP containing protease and phosphatase inhibitors (Roche). Samples
were centrifuged at 21,000 × g for 1 h at 20 °C. The buffer of the solubilized proteins was
exchanged using a PD 10 desalting column equilibrated with PF 2D Start Buffer. Before injection,
samples were centrifuged at 21,000 × g for 1 h at 20 °C. Protein quantification was performed as
stated above. Proteins were separated in the first dimension by chromatofocusing (CF), performed
on an HPCF-1D column. Proteins bound to the strong anion exchanger were eluted with a
continuously decreasing pH (8.5–4.0) gradient generated by start buffer (SB) and eluent buffer
(EB). SB and EB eluent buffer were carefully adjusted to pH 8.5 with 1.0 M NH4OH and to pH 4.0
with a saturated solution (50 mg/ml) of iminodiacetic acid, respectively. Chromatofocusing was
carried out at room temperature, at a flow rate of 0.2 ml/min. Before sample injection, the column
was equilibrated with 50 column volumes of SB until a stable baseline pH (8.5–8.1) was recorded;
20 min after the injection, the elution was started by switching to the eluent buffer at pH 4.0. At the
end of this step 1.0 M NaCl eluted proteins still retained on the column. Fractions were collected
every 0.4 pH interval during the gradient; otherwise fractions were collected on a time basis for a
maximum volume of 1 ml. SDS-PAGE sample buffer (6X) was added to protein samples from each
fraction (50l); samples were boiled for 5 min and subjected to SDS-PAGE. Following
electrophoresis, the gel was transferred to nitrocellulose and immunoblotted as described above.
PKR Kinase Assay. PKR was immunoprecipitated from pre-cleared lysate by adding 1 g of either
-N-ter hPKR or -PKR (D20) and incubating overnight at 40C. Fresh protein A:G agarose beads
(30 l) were added for the final 2 h of immunoprecipitation. Immunoprecipitates were washed three
times in GLB and once with 2X PKR assay buffer (40 mM Tris-HCl, pH 7.6, 4 mM MgCl2, 4 mM
MnCl2). Kinase reaction mix [50 l; 20 mM Tris-HCl, pH 7.6, 2 mM MgCl2, 2 mM MnCl2, 1 mM
DTT, 100 M ATP, 10 Ci -[32P]-ATP (Perkin-Elmer; Monza, Italy), substrate (1 g myelin basic
protein (MBP) or Flag-eIF2)] was added to the immunoprecipitate and incubated at 300C for 30
min. For assays using the PKR inhibitor (PI), the immunoprecipitates were incubated in 2X PKR
kinase buffer with PI for 5 min on ice prior to the addition of substrate, cold ATP and -[32P]-ATP.
Reactions were stopped by the addition of 10 l of 5X protein sample buffer (250 mM Tris-HCl,
pH 6.8, 10% SDS, 2% 2-mercaptoethanol, 0.5% bromophenol blue, 20% glycerol). Samples were
boiled for 5 min and separated by 12% SDS-PAGE. Gels were transferred to nitrocellulose and
exposed to Kodak General Purpose autoradiograph film. Blots were later immunoblotted with -Nter hPKR or -PKR (D20) and in some cases -eIF2 antibody as described above. Change in
activity was determined as a ratio of the change in substrate phosphorylation versus the change in
the amount of PKR protein.
Mass Spectrometry Analysis of Immunoprecipitated Nuclear PKR. Isolated nuclei were lysed
in RIPA buffer containing the appropriate protease and phosphatase inhibitors as described above.
Glycerol was added to 10% of the lysate volume. Lysates (3-5 mg) for each control and test sample
were pre-cleared overnight at 40C by the addition of 3-5 g of non-specific mouse IgG (SCB). The
following morning 30 l of Protein A:G agarose beads were added for the final 2 h of incubation.
The pre-cleared lysates were centrifuged and transferred to a new microfuge tube. Either 3-5 g of
non-specific mouse IgG (control) or -N-ter PKR was added and allowed to complex overnight at
40C. Fresh Protein A:G agarose beads (30-50 l) were added for the final 2 h of the incubation.
Samples were washed four times in IP wash buffer (10 mM Tris-HCl, pH 7.4 and 1% NP-40) and
then resuspended in 40 l IP wash buffer containing 10 l of either reducing or non-reducing 5X
protein sample buffer. Immunoprecipitates were separated by SDS-PAGE, and the gels were then
Coomassie blue stained. Individual lanes were fractionated into 2 mm slices for analysis.
Additionally, a small fraction of the immunoprecipitate (5 l) was separated by SDS-PAGE,
transferred and immunoblotted with either -N-ter PKR or -PKR (D20) as a control for PKR
precipitation.
Gel slices were washed in 100 mM ammonium bicarbonate (pH 8) and 50% acetonitrile
(ACN) until complete destaining and then digested overnight at 370C with sequencing grade trypsin
(Promega, Madison, WI, USA). Peptides were sequentially extracted three times with 50% ACN
and 0.1% formic acid (FA) in water. Each extraction involved 5 min of stirring followed by
centrifugation and removal of the supernatant. Supernatants were combined and completely dried
down. Dried peptide fractions were resuspended in 20 µl of 2% (v/v) ACN, 0.1% (v/v) FA and a 1
µl aliquot, corresponding to 1 pmol/µl was loaded with a l/min flow rate onto a QTOF G6520
(Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a 1200 series capillary pump, a
1200 series nano pump and the Chip Cube system. The LC-Chip (Agilent) was constituted of a
Zorbax 300SB-C18 enrichment column (4 mm x 40 nl, 5 µm) and a Zorbax 300SB-C18 analytical
column (75 mm x 43 mm, 5 µm).
Elution from the analytical column was performed by a binary solvent mixture composed of
3% (v/v) ACN, 0.1% (v/v) FA (solvent A) and 97% (v/v) ACN, 0.1% FA (solvent B) at a flow rate
of 0.4 µl/min with the following gradient: from 4% to 30% B in 17 min, from 30% to 40% B in 3
min, from 40% to 85% B in 3 min, 85% B for 2 min.
Data were acquired in data-dependent MS/MS mode in which, for each cycle, the three most
abundant multiply charged peptides (2+ to 4+) above a threshold of absorbance of 200 in the MS
scan (m/z full scan acquisition range from 250 to 2450) were selected for MS/MS (m/z tandem mass
spectrum acquisition range from 50 to 3200). Each peptide was selected twice and then dynamically
excluded for 0.1 min. For peptide sequence searching, the mass spectra were processed and
analyzed using the Mascot v.2.2 against the UniProt-SwissProt database, allowing for
carbamidomethylation of cysteines and oxidation of methionines with mass tolerances of 20 ppm
for MS and 0.1 Da for MS/MS, allowing for one missed (trypsin) cleavage, and including peptides
with a 5% confidence threshold score.