Ecl prime substrate

Cells/tissues were transferred to RIPA buffer (50 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol). Protein lysates were centrifuged at 12,000× g for 10 min, and the supernatant was collected. Protein concentrations were determined using a Bicinchoninic Acid Protein Assay Kit (Pierce Biotechnology, Waltham, MA, USA). Then, 10 µg of protein lysates per sample were added on a 5–20% gradient of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose membranes (RPN303D, Sigma–Aldrich). Membranes were incubated for 1 min at room temperature (RT) in polyvinyl alcohol and kept in primary antibodies solutions overnight at 4 °C. Membranes were then washed with TBS-Tween 0.1% and incubated with secondary antibodies for 1 h at RT. Membranes were incubated with an ECL Prime substrate (RPN22232, Sigma–Aldrich) for 30 s. Signal detection was performed using the PXI/PXI Touch from Syngene (Synoptics group, Cambridge, UK), blots were quantified with ImageJ™ software. For detailed description of antibodies used (see Supplementary File S1). TIA1 antibodies G-3 and C-20 validation blots can be found in Supplementary File S1.

Cells were lysed with MPER lysis buffer (Life Technologies), centrifuged at 17,740 x g at 4 ˚C for 10 minutes, and protein concentration determined by BCA assay. Equalized samples were loaded onto 4–12% pre-cast polyacrylamide gel (Novex NuPage, Life Technologies), transferred to a Nitrocellulose membrane (Life Technologies), blocked for an hour with 5% skimmed milk dissolved in TBS + 0.05% Tween-20 (TBS-Tw), and then incubated with primary antibodies as indicated in the text in blocking solution overnight at 4˚C. After washing in TBS-Tw, membranes were incubated with appropriate secondary HRP-labelled antibodies (DAKO, Agilent, Santa Clara, U.S.A.) in blocking solution, treated with ECL Prime substrate (Sigma-Aldrich) in accordance with manufacturer’s instructions, and imaged on CL-Xposure film (Thermo-Scientific).