TNFR2 Protein Expression Analysis

Cells were washed, lysed, and processed as previously described (Liu et al., 2020 (link)). Equal amount of proteins for each sample were separated by 10% SDS-polyacrylamide gel and then transferred to Nitrocellulose (NC) membrane for 70 min at 100 V. The membrane was blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline-Tween 20 (0.1%) (TBS-T) for 1hr at room temperature. Rabbit anti-TNFR2 antibody (Abcam, MA, United States, ab109322) were used at 1:1000 dilution at 4°C overnight. β-Actin was detected using anti-rabbit antibody (Abcam, MA, United States, ab179467) as reference protein. Membrane was rinsed with TBS-T for 10 min, five times and incubated with 1:5000 goat anti-rabbit-Alexa Fluor 680 or donkey anti-mouse-Alexa Fluor 680 (Molecular Probes, MA, United States) for 1 h at room temperature. The blots were scanned using an Odyssey fluorescence scanner (LI-COR Biosciences, NE, United States).