Affinipure goat anti rabbit igg h l sa00001 2

Cells were disrupted by RIPA Lysis Buffer (Beyotime) and scraped off the plate surface with a cell scraper. Protein concentration was measured using an enhanced BCA Protein Assay Kit (Beyotime). SDS-PAGE (10%) gels were prepared using SDS-PAGE Gel Quick Preparation Kit (Beyotime). Equal amounts of protein by weight were loaded on the SDS-PAGE and electrophoresed at 80 V for 30 minutes followed by 120 V for 45 minutes. Subsequently, the proteins on the gel were transferred on to polyvinlyidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk for 2 hours, and incubated with primary antibodies overnight at 4°C. Following incubation with the corresponding HRP-conjugated secondary antibody for 1 hour, membranes were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). GAPDH was used as an internal loading control. The following antibodies were used: anti-LIPH antibody (Abcam, ab192615), anti-HIF-1α antibody (36169s; CST; Boston, MA, USA), anti-Slug antibody (12129-1-AP; Proteintech Group), anti-E-cadherin antibody (20874-1-AP; Proteintech Group), anti-N-cadherin antibody (22018-1-AP; Proteintech Group), and HRP-conjugated AffiniPure goat anti-rabbit IgG(H+L) (SA00001-2; Proteintech Group).

Anti-caspase-3 (sc-7272), anti-caspase-4 (sc-56056) and anti-cleaved caspase-4 (sc-22173-R) antibodies (Abs) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-cleaved caspase-3 (ab2302), anti-protein disulfide isomerase (PDI; ab2792) and anti-CCAAT-enhancer-binding protein homologous protein (CHOP; ab11419) Abs were purchased from Abcam (Hong Kong) Ltd. (Hong Kong, China). Anti-β-actin (60008–1-Ig), anti-Bcl-2-associated X protein (Bax; 50599-2-Ig), anti-Bcl-2 (12789-1-AP), anti-cytochrome c (10993-1-AP), peroxidase-conjugated AffiniPure goat anti-mouse immunoglobulin (Ig)G (H+L) (SA00001-1) and peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) (SA00001-2) Abs were purchased from Proteintech Group, Inc. (Chicago, IL, USA). The anti-calpain-1 catalytic subunit (#31038-1) Ab was purchased from Signalway Antibody LLC (College Park, MD, USA). Cisplatin was purchased from Sigma-Aldrich; Merck KgaA (Darmstadt, Germany) and was dissolved in normal saline for in vitro use. TAT-IDP^S was synthesized and purified by ChinaPeptides Co., Ltd. (Shanghai, China) and was dissolved in ddH₂O. The sequence for TAT-IDP^S is RKKRRQRRRGGNVYTEIKCNSLLPLDDIVRV. 2-Aminoethyl diphenylborinate (2-APB; ab120124) was purchased from Abcam (Hong Kong) Ltd.