P1008

Cells were cultured in flask or plate for indicated conditions, and lysed with RIPA buffer (P0013K, Beyotime) containing cocktail protease and phosphatase inhibitors (P1008, Beyotime). Protein concentration was measured by a BCA Protein Assay kit (P0011, Beyotime). Total proteins were separated on 10% reducing SDS PAGE gel and transferred to 0.2 μm PVDF membrane (Merck KGaA, Darmstadt, Germany) for Western blot analysis.

Cells were lysed in RIPA lysis buffer (P10013B, Beyotime) with protease inhibitor (P1008, Beyotime) and phosphatase inhibitor (P1087, Beyotime). Total protein concentration was measured by the Bradford assay and separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, then transferred to polyvinylidene fluoride membrane (Millipore). The membrane was blocked with 5% skimmed milk at room temperature (RT) for 1 h and probed with primary antibodies and subsequently incubated with HRP-conjugated secondary antibodies. The antibodies used in this study are listed as follows. Primary antibodies: anti-Ssrp1 (ab137034, Abcam), anti-GAPDH (HC301, TransGen), anti-β-Tublin (66240-1, Proteintech), anti-phospho-Histone H2A.X (ab11175, Abcam), anti-Spt16 (sc-165987, Santa Cruz). Secondary antibodies: goat anti-mouse IgG-HRP (ab97040, Abcam) and goat anti-rabbit IgG-HRP (ab97051, Abcam).

Grinding adjacent normal tissues and ccRCC tissues were washed in PBS, collected with a cell scraper and ultracentrifuged for 20 min at 10,000 × g at 4°C. The sediments were resuspended in high efficiency cell tissue rapid lysis buffer (RIPA; P0013J, Beyotime, China) with 1% phenylmethanesulfonylfluoride proteinase inhibitors (PMSF; P1008, Beyotime) and 1% phosphatase inhibitors (C071, ChemDiv, USA) inside RIPA. Subsequently, the mixtures were boiled at 95°C for 10 min and then were preserved at −80°C if necessary. Protein quantification were implemented by utilizing the BCA protein assay kit (P0012, Beyotime, China). Total protein mixtures of 20 μg were electrophoresed by direct current in SDS-PAGE gels (LK102, Epizyme Biotech, China) using a Miniprotein III machine (Bio-Rad, USA) and were electro-transferred to PVDF membranes (Millipore, USA) for 2 h, followed by overnight application with primary antibodies targeted CD45RO (5 μg/ml; ab23, Abcam) and GAPDH (1/10000; ab181602, Abcam) at 4°C. Then the membranes were rinsed in triplicates with PBST solution (P0222, Beyotime) and were applied with the secondary goat anti-rabbit IgG antibodies (1:5000; Bioworld Technology, USA) at 20°C for 1 h. Subsequently, the membranes were rinsed again in triplicates and prepared for chemiluminescence by utilizing the Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore).