Gapdh ma5 15738

Cells were washed with PBS and harvested in RIPA lysis buffer (Thermo Fisher Scientific, 89900) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, 87786). Twenty micrograms of cell lysate was denatured in Laemmli SDS-Sample Buffer (Boston BioProducts, BP-110R) and loaded on a precast 4–15% Mini-Protean TGX Stain-Free gel (Bio-Rad). After electrophoresis and transfer, the PVDF membrane (Bio-Rad) was blocked in 5% nonfat milk with TBST (Bio-Rad) at room temperature for 1 h and incubated with primary antibody overnight. MET (3127, 1:1000) antibody was purchased from cell signaling technology. CPD (A305-514A-M, 1:1000) and GAPDH (MA5-15738, 1:5000) antibodies were purchased from Thermo Fisher Scientific. The membranes were then washed in TBST three times, followed by incubation with goat anti-rabbit/Mouse secondary antibodies (Cell Signaling Technology, 1:2000–1:5000) for 1 h at room temperature. After washing with TBST, the membranes were developed by films. All western blots were repeated twice. Image quantification was performed in ImageJ.

From cultured cardiomyocytes, proteins were extracted by adding lysis buffer (Tris 50 mM, NaCl 0.15 M, EDTA 2.5 mM, TRITON X-100 0.2%, IGEPAL 0.3%) to stimulated cells. The protein concentration was estimated by the Bicinchoninic acid assay (BCA Protein Assay Kit, ref 23225, ThermoFisher Scientific). Then, 30–50 ug per sample were loaded on SDS-PAGE gels and separated by electrophoresis. Proteins were then transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The unspecific sites were blocked with skimmed milk in 5% TBST. Primary antibodies were used to quantify the protein levels. Anti-ACADM (ref.: PA5-27201), -PGC-1 alpha (PA5-72948), and -GAPDH (MA5-15738) antibodies were purchased from ThermoFisher Scientific. Anti-Rictor (SAB4200141) was from Sigma and anti-Phospho-AMPKα (Thr172) monoclonal (2535), -AMPKα (2532), -P70S6 Kinase (34475S), and -Phospho-P70S6 (9204S) were from Cell Signaling. The antibody–antigen binding was detected by chemiluminescence. Secondaries antibodies were goat anti-mouse IgG (H + L) (ref: G21040), goat anti-Rabbit IgG (H + L) (ref: G21234), and goat anti-mouse (31430) from ThermoFisher Scientific. Then, antibody complexes were quantified by the ImageJ software v.1.53. All experiments were replicated at least four times.

Chk1 inhibitor MK-8776 was a kind gift from ChemieTek (Indianapolis, IN, USA). Dulbecco’s modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), bovine serum albumin (BSA), antibacterial penicillin/streptomycin solution (100×), trypsin (0.25%) were purchased from Hyclone (GE Healthcare Life Science, Pittsburgh, PA, USA). Monoclonal antibodies, P-gp (C219) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, MA5-15738), and secondary antibody Alexa Fluor 488 conjugated goat anti-mouse IgG used in immunofluorescence assay were purchased from Thermo Fisher Scientific Inc (Rockford, IL, USA), Triton X-100, 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), 36% paraformaldehyde, chemotherapeutics paclitaxel, doxorubicin, colchicine, cisplatin and verapamil were purchased from Sigma-Aldrich (St. Louis, MO, USA). Radioactive [³H]-paclitaxel (15 Ci/mmol) was purchased from Moravek Biochemicals, Inc (Brea, CA, USA). The chemical materials for the ATPase assay were the same as those in our previous work [14 (link),19 (link)].