Anti p glycoprotein antibody

The σ₂ receptor ligand A011 was prepared as previously reported (Feng et al., 2019 (link)). KO143 was purchased from MedChemExpress (Monmouth Junction, NJ, United States). Cisplatin (DDP), adriamycin (ADR), paclitaxel, verapamil, mitoxantrone and rhodamine 123 (Rh123) were acquired from Solarbio (Beijing, China). Cell Counting Kit-8 (CCK-8) was bought from Dojindo Laboratories (Japan). Pgp-Glo™ Assay Systems were purchased from Promega (Madison, USA). Anti-P Glycoprotein antibody, ABCG2, GAPDH, Goat Anti-Rabbit IgG H&L Secondary Antibody (Alexa Fluor 488), Goat Anti-Rabbit IgG H&L Secondary Antibody and Goat Anti-Mouse IgG H&L Secondary Antibody were purchased from Abcam (Cambridge, United Kingdom). SlowFade^TM Gold antifade reagent was purchased from Thermo Fisher (MA, United States).

After the cells were cultured for 24 h after cell seeding, the cells were fixed with PBS containing 4% paraformaldehyde for 10 min and washed with PBS 3 times. Two percent BSA in PBST was then used for blocking for 30 min. After washing 3 times with PBS, the cells were incubated with the Anti-P Glycoprotein antibody (1:100 dilution in 2% BSA in PBST, Abcam, Cambridge, UK) for 1 h at room temperature, followed with incubation with the Alexa Fluor-488 goat anti-rabbit IgG antibody (1:500 in 2% BSA in PBST, Thermo Fisher, Waltham, MA, USA) at room temperature for 1 h in the dark. After 3 times PBS washing, the nuclei were stained with Hoechst 33,258 in the dark for 10 min. The fluorescence images of the stained cells were obtained by a fluorescence microscope.

Protein was collected with 100 μL RIPA lysis buffer with a complete protease inhibitors cocktail (catalog: ab65621, Abcam). An amount of 20 µg protein was loaded on SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were first blocked for 1 h with 5% skimmed milk, then incubated overnight at 4 °C with primary antibodies at a dilution of 1: 1000. The membranes were then rinsed three times with TBS-T and given an additional hour of incubation with HRP-conjugated secondary antibodies at a dilution of 1:5000. The Western Lighting Ultra was used to measure immunoreactivity (Thermo Fisher Scientific). The primary antibodies Anti-P Glycoprotein antibody (catalog: ab216656), Anti-BCRP/ABCG2 antibody (catalog: ab207732), Anti-beta Actin antibody (catalog: ab8226), the secondary antibodies rabbit anti-mouse IgG H&L (HRP) (catalog: ab6728) and goat anti-rabbit IgG H&L (HRP) (catalog: ab6721) were obtained from Abcam.