Traf1

The whole-cell lysates were suspended in lysis buffer containing protease inhibitor (complete mini, Roche), 1 mM PMSF, 1 mM Na₃VO₄ and 1 mM NaF. To attain cytosolic extract and nuclear extract, cells were lysed in hypotonic buffer (10 mM HEPES, PH 7.6, 1.5 mM MgCl₂, 10 mM KCl, 1 mM EDTA, supplemented with protease inhibitor, 1 mM PMSF, 1 mM Na₃VO₄ and 1 mM NaF). Following centrifugation for 5 mins at 3000 rpm, supernatants were continued to centrifugate for 15 mins at 12000 rpm and then supernatants were collected as cytosolic extract; Nuclei-containing pellets were washed 3 times with hypotonic buffer and were lysed in high salt buffer (20 mM HEPES, PH 7.6, 500 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, supplemented with protease inhibitor, 1 mM PMSF, 1 mM Na₃VO₄ and 1 mM NaF). Following centrifugation for 15 mins at 12000 rpm, supernatants were collected as a nuclear extract. Lysates were separated in SDS-PAGE and transferred to polyvinylidene fluoride membrane (Bio-Rad Laboratories). The membrane was incubated with the following primary antibodies: p-IKKα/β, p-IκBα, p65, p50, p-STAT1, STAT1, TRAF1, β-actin, Histone H3 and GAPDH (Cell Signaling Technology) with a dilution of 1:1000.

Nuclear/cytoplasmic fractionation was separated using the Cell Fractionation Kit (Cell Signaling Technology, USA) according to the manufacturer’s instructions, and the whole cell lysates were extracted with RIPA Buffer (Cell Signaling Technology). Western blotting was performed according to a standard method, as described previously [32 (link)]. Antibodies against E-cadherin (Cat# 3195), Vimentin (Cat# 5741), Fibronectin (Cat# 4706), TRAF1 (Cat# 4710), TRAF5 (Cat# 41658) and TRAF6 (Cat# 8028) were purchased from Cell Signaling Technology, p65 (cat# 10745–1-AP) from Proteintech, and p84 (Cat#:PA5–27816) from Invitrogen. The membranes were stripped and reprobed with an anti–α-tubulin antibody (Sigma-Aldrich, USA) as the loading control.