Ecl prime kit

Cells were lyzed with RadioImmunoPrecipitation Assay (RIPA) buffer (Thermo Fisher Scientific, Villebon sur Yvette, France). Cell lysates were clarified by centrifugation at 14000 × g at 4°C for 15 min. For western blotting, proteins were separated by SDS-PAGE gels and transferred to a nitrocellulose membrane. Then, membranes were blocked with Tris buffered saline (TBS) (0.02 M Tris-HCl, 0.137 M NaCl, pH 7.4) containing 0.1% Tween (TBS-T) and 5% non-fat dry milk at room temperature during 1 hour and incubated overnight at 4°C with the following primary antibodies: anti-phospho-JAK2 (Santa Cruz Biotechnology), anti-GAPDH, anti-DDR1, anti-phospho-SHP2, anti-SHP2, anti-JAK2, anti-phospho-ERK1/2, anti-ERK1/2, anti-p21^CIP1 (Cell signaling Technology, Saint Quentin Yvelines, France), anti-DDR2 (R&D systems, Lille, France), anti-RAGE (GeneTex, Irvine, CA). Membranes were washed with TBS-T and incubated with the corresponding peroxidase conjugated secondary antibody at room temperature for 1 hour. Chemiluminescent detection was realized by using an ECL Prime Kit (GE Healthcare, Orsay, France).

Frozen tissues were powdered under liquid nitrogen with the Multi-beads shocker and resuspended in 6 mol/L urea, 2 mol/L thiourea, 3% CHAPS, and 1% Triton X-100. The supernatant was then cleared by centrifugation at 15,000 rpm for 30 min. Cultured cells were washed with PBS, fixed with 10% TCA for 30 min, and lysed in the same buffer for 30 min. The supernatant was collected after centrifugation for 30 min.
Proteins were separated on SDS-polyacrylamide gels and transferred to a nitrocellulose membrane (Bio-Rad). After blocking for 1 h in Tris-buffered saline and Tween 20 (TBS-T) supplemented with 5% nonfat milk, membranes were incubated overnight at 4°C with primary antibodies against Bak1 (monoclonal, 1 : 250, MBL), PARP (1 : 1000, BD), Caspase-3 (1 : 1000, BD), Caspase-9 (1 : 1000, MBL), Caspase-2 (1 : 1000, BD), BMF (polyclonal, 1 : 1000, MBL), PUMA (monoclonal, 1 : 1000, BD), Bcl-2 (monoclonal, 1 : 500, BD), PP2A-catalytic α (1 : 1000, BD), MCL-1 (1 : 1000, BD), and β-actin (1 : 1000, BD). Membranes were then extensively washed with TBS-T and labeled with horseradish peroxidase-conjugated secondary anti-mouse (1 : 1000, GE) or anti-rabbit IgG (1 : 2000, GE). After additional washes with TBS-T, antigen-antibody complexes were visualized with ECL-Prime Kit (GE).

Protein samples were separated by SDS-PAGE in 6% gel or 4–20% polyacrylamide gradient gel, soaked in transfer buffer and electroblotted onto PVDF membrane overnight. The membrane was incubated with primary Abs for 90 min and with HRP conjugated secondary Abs for 30 min at room temperature. Protein bands were visualized with an ECL Prime kit (GE Healthcare UK, Ltd.). For developmental analysis of MAP 1B, the whole brains of E13, E17, postnatal day (P) 2, P8, P15 and adult mice were homogenized in lysis buffer with protease inhibitors and phosphatase inhibitors and centrifuged at 12,000×g at 4 °C. Half volume of 3 × sample buffer for SDS-PAGE was added to each supernatant. To analyze the expression or the phosphorylation level of MAP 1B, ImageJ software Fiji (http://rsb.info.nih.gov/ij) was used to measure the areas under the curve of the immunoblotted bands. In the developmental analysis, the values were normalized to α-tubulin Ab and compared to that of adult brain, which was defined as 1.0.

RIPA buffer (50 mM HEPES KOH pH 7.9, 150 mM NaCl, 1.5 mM MgCl₂, and 1.0% v/v NP-40) was added to cells or tumors for protein extraction. Next, 20 µg of protein were separated on 4–12% Bis-Tris Gels (Life Technologies) and transferred to an Immun-Blot PVDF Membrane for Protein Blotting (BIO-RAD, Hercules, CA, USA). Membranes were incubated with the following primary antibodies: anti-beta-actin (Chemicon, Temecula, CA, USA) and anti-PKR (product number: 3210; Cell Signaling, Danvers, MA, USA). Anti-phosphorylated PKR was purchased from Life Technologies (product number: 44–668 G, Figs. 1 and 2), Abcam (Cambridge, England, product number: ab32036; Figs. S2 and S3). Species-specific secondary antibody kits were purchased from General Electric (Charles Coffin, NY, USA). The signal was visualized with an ECL Prime Kit (General Electric). The density of the bands was quantified by normalization to β-actin using Image J Software (National Institutes of Health, Bethesda, MD, USA).

Intact nuclei and cytosolic fractions were separated by employing the Nuclei EZ Prep kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions. Pure nuclei were extracted from cellular lysates with nuclear extraction buffer (50 mM Tris, pH 8.0, 350 mM NaCl, 1 mM DTT, protease and phosphatase inhibitors (5 mM MgCl₂ and 50 units of DNAse I), and incubating for 2 h at 4 °C [14 (link)]. Total protein in the clarified nuclear lysate was quantified using a Bradford protein assay (Bio-Rad, Hercules, CA, USA; Cat. No. 5000006). Moreover, 25–50 µg of total protein from the nuclear extract was separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane (0.45 μM) overnight. The membranes were then blocked with 5% nonfat dry milk (NFDM) in TBST (20 mM Tris, pH 7.6, 100 mM NaCl, 0.1% Tween-20) for 1 h. Subsequently, the membranes were rinsed with TBST and incubated with primary antibodies in 5% BSA or NFDM for 2 h at 4 °C. Subsequently, the membranes were washed thrice with TBST, and incubated with secondary antibodies (1:5000), conjugated with horseradish peroxidase (HRP) in 1% BSA or NFDM in TBST for 1 h at R.T. The membranes were washed thrice with TBST, and the proteins were detected by enhanced chemiluminescence (ECL) Prime kit (GE Healthcare Life Sciences; Cat. No. RPN2232,).

For Western blot analysis, the cells were rinsed two times with phosphate buffered saline and lysed in SDS electrophoresis sample buffer. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked for 1 h at room temperature with 5% nonfat milk in TBS (10 mM Tris-HCl, pH 7.6, and 150 mM NaCl) and incubated overnight at 4 °C in TBS-T (10 mM Tris-HCl, pH 7.6, and 150 mM NaCl, 0.05% Tween 20) supplemented with BSA (5%) and appropriate antibodies. The membrane was washed three times with TBS-T, incubated for 1 h with 5% nonfat milk in TBS-T and horseradish peroxidase conjugated goat anti-rabbit IgG (1:4000, 7074, Cell Signaling Technology, Danvers, MA, USA) and then washed three times with TBS-T. Immunocomplexes were detected using an ECL Prime kit (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s recommendations. The rabbit monoclonal primary antibodies were from Abcam (ACTB, ab8227, Cambridge, UK) and Cell Signaling Technology: phospho-eIF2 S51 (D9G8), phospho-p70 S6 Kinase T389 (108D2), phospho-S6 ribosomal protein S235/236 (D57.2.2E), S6 ribosomal protein (5G10), phospho-4EBP1 T37/46 (236B4), 4EBP1 (53H11), histone H3 (D1H2). Histone H3 antibodies were used as 1:10000 dilution, other primary antibodies were used as 1:2000 dilution.