Gadph antibody

Oxaliplatin (L-OHP, Jiangsu Hengrui, 50mg/pc), doxorubicin hydrochloride (ADM, Zhejiang Haizheng, 10mg/pc), 5-fluorouracil (5-FU, Jiangsu Nantong Jinghua Pharmacy, 0.25g/10ml), and VER (Verapamil, VER, Shanghai Hefeng Pharmacy, 5mg/2ml) were supplied by our hospital. The CCK-8 kit was purchased from Japan Dojindo Laboratories. The RNA extraction and reverse transcription kit were purchased from TIANGEN. 2×SYBR Green universal qPCR Master Mix was purchased from TIANGEN. Mouse-anti human CDKN2B/P15 and P-gP primary antibody were purchased from Abcam. The GADPH antibody was purchased from Sigma. The second antibody labeled by sheep-anti mouse HRP was purchased from Guizhou Jinqiao Biotechnology. The high-flux sequencing was performed by Guangzhou Ruibo. siRNA for gene transfection was purchased from Guizhou Ruibo. The overexpression plasmid and empty carrier TrueORF GOLD type were purchased from ORIGENE. The annexin V-PI double-staining kit was purchased from Beijing Beibo. The Lipofectamine 3000 was purchased from Invitrogen. Primer design and synthesis were performed by Shanghai Shanjing Biotechnology Company.

Snap frozen hearts were used to quantify protein expression of TGF-β1 as previously described [13 (link)]. Fifty µg protein samples were separated by 10% SDS-PAGE gels, and the primary antibody against TGF-β1 (rabbit polyclonal Santa Cruz Biotechnology; 1:1000 dilution; Heidelberg, Germany) was applied overnight at 4 °C. Thereafter, the primary antibody was washed, and the secondary antibody (anti-rabbit IgG-peroxidase conjugated, dilution 1:10000) was applied for 1 h at 37 °C. The blots were then washed and incubated in commercial enhanced chemiluminescence reagents (ECL Prime, Amersham Biosciences, Buckinghamshire, UK), and the bands were detected by the ChemiDoc XRS + Imaging System (Bio-Rad, Hércules, CA, USA). To prove equal loadings of samples, blots were re-incubated with the GADPH antibody (1:3000 dilution; Sigma-Aldrich, Hércules, CA, USA). Blots were quantified using Image Lab 6.1 software (Bio-Rad, Hércules, CA, USA), and expression values were normalized with GADPH.