Western blot chemiluminescence reagent plus

Western blotting was performed as described^{17 (link), 29 (link)}. The cell lysates or pulled down complexes were subjected to SDS-polyacrylamide gel electrophoresis for western blotting analyses with antibodies against β-actin (Millipore Merck Chemicon, Pittsburgh, PA); G3BP1 (Santa Cruz); p65, p-p65 (S536), IκBα, p-IBα (S32), p-IKKα/β (S180/181), p-Stat3 (Y705), Stat3, JAK1 (Y1022/1023), JAK1, p-JAK2 (Y1007/1008), JAK2, p-JAK3 (Y980/981), JAK3, Tyk2, p-Tyk2 (Y1054/Y1055), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology); Caprin-1 (Proteintech Group) and horseradish peroxidase-conjugated secondary antibodies (PerkinElmer). Enhanced chemiluminescence detection reagents (Western Blot Chemiluminescence Reagent Plus; PerkinElmer) were used according to the manufacturers’ instructions to detect antigen–antibody complexes.

Cells were harvested, washed in PBS, and then lysed on ice for 30 min in RIPA buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA pH 8, 1% NP-40, 0.5% Na-deoxylcholate and 0.5% SDS supplemented with complete EDTA-free protease inhibitor (11697498001; Roche, Mannheim, Germany). Cellular debris was removed by centrifugation, and protein concentration in cell lysates was measured by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific), according to manufacturer’s instructions. SDS-PAGE analysis was performed on 50 µg of total protein extract. After separation, proteins were transferred to Hybond-P membrane (GE Healthcare, Freiburg, Germany). Immunoblotting was carried out with the following antibodies: mouse monoclonal anti-vinculin (sc-25336; Santa Cruz Biotechnology, Heidelberg, Germany) and rabbit monoclonal CAV-1 (D46G3; Cell Signalling Technology) at 1:1000 dilution. After incubation with horseradish peroxidase conjugated secondary antibodies (Santa Cruz Biotechnology) at 1:1000 dilution, proteins were revealed with Western Blot Chemiluminescence Reagent Plus (Perkin Elmer Life Sciences) and exposed to Hyperfilm™ ECL radiographic films (GE Healthcare).