Ab109322

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).

RIPA cell lysis buffer (Solarboi, China), containing 1% PMSF and 1% phosphatase inhibitor cocktail (Cwbio, China), was used to cleave proteins for 15 mins on ice. The lysed cells were centrifuged at 12000 rpm for 15 min, and the supernatants were collected for protein quantification using a BCA Protein Assay kit (Beyotime, China). The total protein was separated by 7.5% SDS-PAGE (NCM Biotech, China) and transferred to 0.45-μm PVDF membranes (Millipore, Burlington, MA, USA). The membranes were washed with blocking solution (Yoche, Shanghai, China) for 10 mins and incubated on a shaker at 4°C overnight with diluted TNFR2 (1:1000, ab109322, Abcam, UK), β-Tubulin, (1:1000, A12289, ABclonal, China), phospho-IKK alpha/beta (1:1000, AF3013, affinity), phospho-IκBα (1:1000, AP0707, ABclonal, China), NF-κB (1:1000, A19653, ABclonal, China), phospho-NF-κB(1:1000, AP0124, ABclonal, China). Membranes were washed in 0.1% Tris-buffered saline Tween-20 and incubated in goat anti-rabbit IgG H&L (HRP) (1:2,000, LF102; Epizyme, China) for two hours. Finally, protein bands were visualized using WesternBright ECL (Apgbio, China) and quantified using Image J.

Immunohistochemistry was performed in formalin-fixed, paraffin-embedded kidney sections from SS rats (n = 6–9), following procedures similar to what we described previously33 (link)34 (link)35 (link). The primary antibodies, all from Abcam, and the dilution used were ab6671 for TNFα, 1:100, ab19139 for TNF receptor I, 1:1500, and ab109322 for TNF receptor II, 1:100.