Abcg2 antibody

Cell lysis was performed with Invitrogen cell extraction buffer (FNN0011) after washing with DPBS. Whole protein quantification was determined by using BCA protein assay reagents (Pierce™ BCA Protein Assay Kit). SDS-PAGE was performed by using Mini-PROTEAN TGX stain-free Gels from BioRad (Feldkirchen, Germany) after dilution of lysates with Laemmli sample buffer (BioRad #1610747) and denaturation for 30–45 min. PVDF membranes were incubated with respective antibody and mouse anti-GAPDH (Affinity Biosciences; #T0004; 1:10,000 dilution). Western blots were quantified via chemiluminescence using Clarity Western ECL Substrate (BioRad#1705061). Detection was performed with the ChemiDoc XRS+ imaging acquiring system (BioRad) and Image Lab software v. 6.0.1 (BioRad). Antibody Thrombin R (ATAP2) (Santa Cruz biotechnology; sc-13503; 1:200 dilution) was used to detect PAR-1 expression. For BCRP detection, we used ABCG2 antibody (Santa Cruz biotechnology; sc-58222; 1:200 dilution). As a secondary antibody, we used HRP-conjugated anti-mouse IgGκ binding protein (Santa Cruz biotechnology; sc-516102; 1:10,000 dilution).

Antibodies against GAPDH, EpCAM (D9S3P), AKT, P-AKT(S473), mTOR, P-mTOR, S6K, P-S6K(T398), 4EBP1, P-4EBP1, PTEN, CD44, Oct4, Nanog, c-myc, cyclin D1 and Slug were purchased from Cell Signaling Technology. ABCG2 antibody was obtained from Santa Cruz Biotechnology. Mouse monoclonal antibodies against human α-catenin, E-cadherin, fibronectin and Vimentin were purchased from BD Biosciences. GAPDH antibody was used for a normalised control. Rapamycin and the AKT inhibitor MK2206 were purchased from Selleck Chemicals. To test the effect of AKT/mTOR activation on cell invasiveness, sphere formation ability and gene expression induced by EpCAM expression, NPC cells were pretreated with the AKT inhibitor MK2206 (5 μM) or Rapamycin (100 nM) for 24 h before performing the following experiments and assays.

Cells were lysated with 1×RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) and analyzed by western blot assay. Primary antibodies including ABCG2 antibody (Cat No. 58222), ALDH1 antibody (Cat No. 166362) and GAPDH (Cat No. 25778) were all from Santa Cruz Biotechnology (Dallas, Texas, USA). Secondary anti-mouse or anti-rabbit IgG were supplied by Zhongshanjinqiao Biotech (Beijing, China), and the final signal was detected using the UVP Imaging.