Gapdh antibody

Pyrrolidine dithiocarbamate (PDTC) was purchased from Sigma-Aldrich. TLR4 antibody was obtained from Bioss (USA), NF-κB antibody was bought from Abcam (USA), and GAPDH antibody was from ZSGB-BIO (China).

Western blotting was performed in accordance with the standard protocols. In brief, cellular proteins were extracted using the RIPA lysis buffer, separated on SDS-PAGE gels, and transferred onto NC membranes (Millipore, Billerica, USA). After incubated in primary and second antibodies separately, proteins were detected by ECL reagent (Millipore, USA) and imaged with FluorChem Q (Protein simple, USA). The following antibodies were used for western blotting: PKM2 antibody (Proteintech, China, 1 : 1000, 15822-1-AP), vimentin antibody (Proteintech, China, 1 : 1000, 10366-1-AP), E-cadherin antibody (Proteintech, China, 1 : 1000, 20874-1-AP), N-cadherin antibody (Proteintech, China, 1 : 1000, 22018-1-AP), and GAPDH antibody (ZSGB-bio, China, 1 : 1000, TA-8).

RIRA lysate buff was used to extract total protein from renal cancer cell lines (A498, 786-O, ACHN, Caki-1, and OSRC-2) and normal renal tubular epithelial cells (HK-2), and four pairs of ccRCC tissues and matched adjacent normal tissues from our study centre. Protein concentrations were determined by the BCA method using a bicinchoninic acid kit (CoWin Biosciences). Then, 30ug of total protein was separated by 10% SDS-PAGE (Servicebio, China) and transferred to PVDF membranes (0.45 mm,Immobilon-P Transfer Membrane). The membrane was subsequently blocked for 1 h at room temperature using 5% skim milk and inclubated with corresponding DBT antibody (Affinity Biosciences,DF13569,Rat, 1:1,000) or GAPDH antibody (ZSGB-BIO,TA-08,Mouse, 1:1,000) overnight at 4°C. After rinsing with TBST (Servicebio, China), the membrane was placed in the corresponding secondary antibodies and incubated for 1 h at room temperature, and rinsed again with TBST (Servicebio, China), and target protein expression levels were detected with an ECL kit (Fdbio science, China,Hangzhou) and a DNR Bioimaging System, analysed by using GraphPad Prism 9.