Anti gapdh ap0063

Cells were lysed in RAPI (Sigma-Aldrich, St. Louis, MO, USA) containing 1% phenylmethylsulfony fluoride (Beyotime, Shanghai, China) and centrifuged for 15 min at 4℃. Protein concentrations were then measured using a BCA protein assay kit (Beyotime). Proteins were separated on 12% gels using sodium dodecyl sulfate polyacrylamide gel electrophoresis. For immunoblots, the PVDF membrane carrying the transferred proteins was incubated at 4℃ overnight with designated primary antibodies diluted in TBST buffer containing 0.1% Tween20 and 5% bovine serum albumen (BSA). Immunodetection was accomplished using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection system (P1108, Beyotime). The antibodies anti-CDK2 (ab32147), anti-HSP90 (ab13492), anti-Cdc37 (ab224831), and anti-ubiquitin (ab134953) were from Abcam, and anti-GAPDH (AP0063) was from Bioworld Technology Inc. (St. Louis Park, MN, USA). Images were analyzed using ImageJ. (National Institutes of Health, Bethesda, MD, USA).

The following antibodies were purchased: anti-BRD4 (E2A7X) from Cell Signaling Technology, anti-BRD2 (D89B4) from Cell Signaling Technology, anti-BRD3 (2088C30) from Abcam Technology, anti-c-Myc (A19032) from ABclonal Technology, anti-c-Met (25869-1-AP) from Proteintech Technology, anti-EGFR (2232) from Cell Signaling Technology, and anti-GAPDH (AP0063) from Bioworld Technology Inc. All antibodies were utilized at 1:1000 dilutions, except GAPDH (1:10,000). JQ1 (HY-13030) and (2-Hydroxypropyl)-β-cyclodextrin (HY-101103) were purchased from Haoyuan Chemexpress Co., Ltd. Trichostatin A (T6270) was purchased from Target Mol, Inc. Vorinostat (T1583) was purchased from Target Mol, Inc. Osimertinib (HY-15772) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Reagents were dissolved in DMSO at 20 mmol/L, stored at -20℃, and diluted just before use. Lipofectamine 2000® transfection reagent (11668-019) was purchased from Life Technologies Co. Invitrogen (Carlsbad, CA, USA).

Western blotting was performed according to standard methods. Antibodies of CCDC65 (24376-1-AP), ENO1 (11204-1-AP), c-Myc (10828-1-AP), CCND1 (60186-1-Ig), p-AKT (Ser473) (66444-1-Ig), DYKDDDDK Tag (80010-1-RR), mTOR(66888-1-Ig), p-mTOR(Ser2448) (677778-1-Ig), GSK3β(22104-1-AP), p-GSK3β(Ser9) (67558-1-Ig), were purchased from Proteintech (Rosemont, IL, USA). Anti-P-Akt1 (Ser473) (#9018), Mouse IgG (#3420), normal Rabbit IgG (#2729), Anti-p27(#3686), Anti-p21(#2947) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-AKT1 (48884) was purchased from Signalway Antibody (College Park, MD, USA). Anti-flag (F1804) was purchased from Sigma (St. Louis, MO, USA); Anti-β-actin (AP0060) and anti-GAPDH (AP0063) were purchased from Bioworld (Bloomington, MN, USA). Antibodies were diluted with SuperKine™ Enhanced Antibody Dilution Buffer (Abbkine, Wuhan, China). Western blotting was performed by Mini-PROTEAN Tetra- and Mini Trans-Blot (Bio-Rad, Hercules, CA, USA). The images were captured with Chemiluminescence Imaging System (Minichemi, Beijing, China).

The 6 dpi or sham myocardial tissue was lysed with lysis buffer containing 0.1% protease inhibitor, 1% phosphatase inhibitor, and 0.5% phenylmethylsulfonylfluoride (GeneChem, Shanghai, China). After 20 g of protein from each sample was separated on 10% SDS-PAGE gels (EpiZyme Scientific, Shanghai, China) and the separated proteins were transferred onto polyvinylidene fluoride membranes (Roche Applied Science, Mannheim, Germany), 5% BSA (Solarbio Science & Technology Co., Ltd, Beijing, China) was used to block the membranes. Then, the blocked strips were incubated with primary antibodies overnight at 4 ^∘C. The primary antibodies used were as follows: anti-Gpx3 (ab256470, 1:1000, Abcam), anti-Ankrd1 (11427-1-AP, 1:4000, Proteintech), anti-Trim72 (22151-1-AP, 1:4000, Proteintech), anti-GAPDH (AP0063, 1:5000, Bioworld), and anti-Tubulin (AP0064, 1:5000, Bioworld). After the membranes were cultured with an HRP-linked anti-rabbit IgG Antibody (1:3000, Cell Signaling Technology, Inc.) at room temperature for 2 h, the proteins on the membranes were visualized with enhanced chemiluminescence (ECL) reagents (FDbio, Hangzhou, China) on an iBright FL1000 Imaging System (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The band intensity was quantified by ImageJ software.