Antibody for gapdh

TSE₁ (Figure 7) was purchased from Yingshili Biotechnology (Hangzhou, Zhejiang, China), and was dissolved in dimethyl sulfoxide (DMSO) at 100 mM and stored at −20 °C PLT, 6-well plates, 24-well plates, 96-well plates, Transwell chambers and Corning Matrigel Basement Membrane Matrix High Concentration were from Corning (Corning, NY, USA). Aqueous One Solution Cell Proliferation assay (MTS) was purchased from Promega Corporation (Madison, WI, USA). RPMI-1640 medium was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was from Invitrogen (Grand Island, NY, USA). ALDEFLUOR Stem Cell Identification Kit, MammoCult Medium Human Kit, Hydrocortisone Stock Solution, 0.2% Heparin Solution and HBSS were obtained from Stem Cell Technologies (Vancouver, BC, Canada). Primary antibodies for Oct-4, CD44, Nanog, Bcl-2, Notch-1, MMP-9 and horseradish peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibody for GAPDH was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

The cultured cells were washed with cold phosphate buffer saline (PBS) three times and harvested using a cell lysis buffer containing protease inhibitors PMSF (Solarbio Life Sciences, Beijing, CHN). Equal amounts of protein from control and treated cell lysates were separated using a 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel under reducing conditions and transferred onto polyvinylidene fluoride (PVDF) membranes (Solarbio Life Sciences, Beijing, China), which were subsequently probed with primary antibodies (AChRα9, Santa Cruz, sc-293282, CA, USA). In all Western blots, membranes were additionally probed with an antibody for GAPDH (Santa Cruz, sc-47724, CA, USA) to ensure equal loading of protein between samples. Horseradish peroxidase-conjugated secondary antibodies (m-IgGκ BP-HRP, Santa Cruz, sc-516102, CA, USA) were used with enhanced chemoluminescence reagent (Biosharp, Guangzhou, China) to visualize the protein bands. Images of the films were captured using the Alpha FluorChem E (ProteinSimple, CA, USA).

Preparation of cell lysates and Western blotting were performed as described previously [22 (link)]. Antibodies for PARP, cleaved PARP, caspase 3, caspase 9, RIP1, MLKL, Beclin, p62, and peroxidase-conjugated secondary antibody were purchased from Cell Signaling (Danvers, MA). Antibody for GAPDH was purchased from Santa Cruz Biotechnology (Dallas, TX). The blotting membranes were probed with 1 : 1000 diluted primary antibody and 1 : 4000 for the peroxidase-conjugated secondary antibody. Immune complexes were detected by chemiluminescence using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Grand Island, NY) and photographed using myECL imager instrument (Thermo Fisher Scientific, Grand Island, NY).

For Western blot analysis, polypeptides in whole cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membrane filters. Detection was performed with a 1:5000 or 1:10,000 dilution of primary antibody using an enhanced chemiluminescence (ECL) system. Images were acquired using the LAS4100 system (GE Healthcare, Uppsala, Sweden). The antibodies for p62 and LC3 were purchased from MBL (Woburn, MA, USA), antibody for PARP from GeneTex (San Antonio, TX, USA), that for actin from Applied Biological Materials (Richmond, Canada), and the antibody for GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA, USA).