Protease inhibitor cocktail

Cells were lysed in cell lysis buffer (cell signaling technology, USA) with a protease inhibitor cocktail (BBI Life Sciences, China). Proteins were separated by 10% or 12% SDS-PAGE and transferred onto PVDF membranes. After blocking in 5% non-fat milk, the membranes were incubated with primary antibodies overnight at 4°C and detected by incubating with specific secondary antibodies (Jackson ImmunoResearch, USA) for 2 h at room temperature. Then protein bands were exposed with ECL chromogenic substrate. β-Actin (Huaan biotech, China) served as an internal loading control. Other primary anti-human antibodies include UCH-L5 (Santa Cruz, USA), SNRPF (abcam, USA), Flag-tag (MultiSciences Biotech, China) and HA-tag (Abmart, China).

The cells were harvested, washed, and lysed with RIPA lysis buffer containing a protease inhibitor cocktail (BBI Life Sciences, Shanghai, China) together with NaF and Na₄P₂O₇, and the protein concentration was measured using a BCA Protein Assay Kit (Pierce, Waltham, MA, USA). Subsequently, the samples were loaded onto SDS-PAGE gels and separated through electrophoresis. The proteins were transferred onto PVDF membranes and incubated with the corresponding primary Abs and HRP-conjugated secondary Abs. Chemiluminescent detection was performed using Alliance 4.7 (UVITEC Cambridge, Cambridge, UK) with Luminata Forte Western HRP Substrate (Millipore, Billerica, MA, USA).
The following antibodies were used: Phospho-IκBα (Ser32) (CST#2859, Cell Signaling Technology, Danvers, MA, USA), IκBα (CST#4812), Phospho-NF-κB p65 (Ser536) (CST#3033), NF-κB p65 (CST#3034), NIK (CST#4994), NF-κB2 p100/p52 (CST#4882), RelB (CST#4922), Phospho-Stat3 (Tyr705) (CST#9131), Phospho-Stat3 (Ser727) (CST#9134), SHP-1 (CST#3759), SHP-2 (CST#3397), Stat-3 (ab68153, Abcam, Cambridge, UK), β-Actin (ACTB) (BM0627, Boster, Wuhan, China), HRP Anti-rabbit IgG (CST#7074), and HRP anti-mouse IgG (BioLegend, 405306).