Bolt lds buffer

Samples were collected with Bolt LDS Buffer and Bolt Reducing Agent (Invitrogen, Waltham, MA, USA) and run on polyacrylamide gels. Gels were transferred using the iBlot 2 Gel Transfer Device (Invitrogen). Membranes were probed with primary antibodies for eIF4G, (1:1000, Santa Cruz Biotechnology), actin (1:2000, ProteinTech, Rosemont, IL, USA), SAT1 (1:100, Santa Cruz Biotechnology), and GAPDH (1:1000, Santa Cruz Biotechnology). Membranes were treated with SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific) and visualized on FluorChem E imager (Protein Simple, San Jose, CA, USA).

VSMC WT cells were lysed for 30 min in co-IP buffer [1% NP-40 (Bio-Shop, NON505), 150 mM NaCl (Sigma-Aldrich, S9888), 1 mM EDTA (TFS, 15575-038), 50 mM Tris HCl pH 7.5 (Invitrogen, 15567027) 10% Glycerol (Bio-Shop, GLY001)] supplemented with protease inhibitors. Cell lysates were immunoprecipitated with IRF1 (CST, 8478, D5E4) and IRF9 (CST, 28845, D9I5H) antibodies overnight at 4°C. Immunocomplexes were isolated with Dynabeads Protein A/G [TFS, 10008D(A), 10009D(G)] saturated with 1% BSA (Sigma-Aldrich, A3059), by gentle rocking for 3 h at. Beads were washed 3 times with ice-cold co-IP buffer and once with Tris-EDTA buffer. Next bound proteins were retrieved by boiling in Bolt LDS buffer (Invitrogen, B0008) for 10 min. Immunocomplexes were analyzed by Western blot (described in Materials and Methods section, Western blot) with tSTAT1 (CST, 14994, D1K9Y) 1:500 and tSTAT2 (CST, 72604, D9J7L) 1:400.