Tnfr1

Immunohistochemistry (IHC) was conducted as previously described [15 (link)]. Tissues were deparaffinized, rehydrated, and incubated at room temperature in 0.3% H₂O₂ to block endogenous peroxidase and in blocking solution for nonspecific binding. Primary antibody were applied to sections overnight at 4°C. Afterwards, tissues were incubated with anti-mouse HRP conjugated (Abcam, USA) secondary antibody for 1 h at room temperature. Then enzyme development was performed with DAB/H₂O₂ complex for 10 min at room temperature and in the absence of light which provides a brownish precipitation. The primary antibodies specific for Fas, Fasl (epitomics, USA) ,TNF-α and TNFR1(Proteintech group,USA) were used at working dilution 1:50, 1:100, 1:50 and 1:200 respectively. Stained (brown) cells were quantified as number of positive cells. To evaluate the intensity of antigen immunoreactivity we examined the percent of positive staining urothelial cells in 10 fields at random per rat per antibody under 400 × magnification.

Recombinant human TNFα (AF-300-01A) was purchased from PeproTech. Cisplatin (HY-17394) was from MedChemExpress, QNZ (S4902) and Cycloheximide (CHX) was from Selleck. The primary antibodies used in this work are as follow: KIAA1522 (WB, 1:1000; IHC, 1:200; HPA032050, Sigma ImmunoChemicals, St Louis, MO, USA), Phospho-NF-kB p65 (Ser536) (WB, 1:1000; CST, 3033); NF-κB p65 (WB, 1:1000; CST, 8242), Phospho-IkBα (Ser32) (WB, 1:1000; CST, 2859), IkBα (WB, 1:1000; CST, 4814), Phospho-IKKα/β (Ser176/180) (WB, 1:1000; IHC, 1:150; CST, 2697), IKKα (WB, 1:1000, CST, 11930), TNFR1 (WB, 1:1000, Proteintech, 21,574–1-AP), TNFR2 (WB, 1:1000; IHC, 1:50, Proteintech, 19,272–1-AP), GAPDH (WB, 1:1000, Santa Cruz, sc-25,778), β-actin (WB, 1:2000, Santa Cruz, sc-47,778), Ki67 (IHC, 1:200, GeneTex, GTX16667), cleaved caspase-3 (IHC, 1: 500, CST, 9664).

After various treatments as indicated, cancer cells were harvested, and the total protein was lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and then quantified by a BCA kit (Epizyme, Shanghai, China). Equal amounts of proteins were resolved on 10%-12.5% SDS-PAGE gels, followed by transferring the proteins to an Immobilon PVDF Membrane (Merck Millipore Ltd, Tullagreen, Ireland). PVDF membranes with proteins were blocked with 5% skim milk for 1 hour, incubated with primary antibodies overnight at 4°C, and then conjugated with secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 hour. Signals were visualized by ECL reagent (Epizyme, Shanghai, China) and photographed by Tanon 5200 visualizer (Tanon, Shanghai, China).
The primary antibodies used are as follows: p27, p21, Cyclin D1, PARP, cleaved PARP (c-PARP), cleaved caspase 3, cleaved caspase 9, cleaved caspase 7, cleaved caspase 8, Noxa, Bak, Bax, Bik, Bim, Bad, Puma, Bcl2, Bcl-xl, Mcl-1, XIAP, BID, TRAIL, DR4, DR5, CHOP, ATF4, eIF2α, and p-eIF2α were from Cell Signaling Technology (USA). Cyclin E, CDK2, CDK4, CDK6, Fas, DR3, c-Myc, and p53 were from Santa Cruz Biotechnology (USA). TNFR1 and TNFR2 were from Proteintech (USA). β-Actin was from HuaBio (China).