β actin polyclonal antibody

The tumor tissue (10 mg) was boiled in a Western blotting (WB) sample buffer for 30 min. The proteins in the WB samples were separated by gel electrophoresis (80 V for 30 min followed by 120 V for 1 h) and transferred to a PVDF membrane (100 V for 40 min). The proteins were incubated with a PD-L1 monoclonal antibody (Cat# 66248–1-Ig, ProteinTec Chicago, IL, USA) [23 (link)] or β-actin polyclonal antibody (Cat# 20536-1-AP, ProteinTech Chicago, IL, USA) [24 (link)] and secondary antibody (Cat# SA00001-1, ProteinTech Chicago, IL, USA) [25 (link)], and the expressions of the mouse PD-L1 and actin were analyzed using a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).

Plates were rinsed twice with PBS before harvesting of the adherent cells. The collected cells were lysed with 1 mL of radioimmunoprecipitation assay (RIPA) lysis buffer (R0010; Solarbio, China) supplemented with 1 mM phenylmethyl sulfonyl fluoride (PMSF), 10 mM dithiothreitol (DTT), 40 μg/mL DNase I, and 1 μg/mL leupeptin, pepstatin, and aprotinin for 30 min on ice. Cell lysis was performed by centrifugation at 12,000 rpm for 30 min at 4°C. The supernatant was collected, and the protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit. Equal amounts of protein were mixed with 1× SDS-PAGE loading buffer, boiled, and subjected to SDS-PAGE. The proteins in the gel were transferred onto 0.45-μm-pore polyvinylidene difluoride (PVDF) membranes and detected with specific or tagged antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. ImageJ was used to quantify the relative band intensities. The following antibodies were used: GFP-tagged monoclonal antibody (66002-1; Proteintech, IL, USA), Flag-tagged polyclonal antibody (20543-1; Proteintech), β-tubulin polyclonal antibody (10068-1; Proteintech), β-actin polyclonal antibody (20536-1; Proteintech), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1; Proteintech), and ABHD16A/BAT5 polyclonal antibody (SRP08788; Saierbio, China).