Akt rabbit polyclonal antibody

Western Blotting assay was used to examine the protein expression as previously described. For the cell derived samples, RIPA buffer containing a protease inhibitor (Sigma) was used to extract the total protein. For nuclei derived samples, the proteins were extracted and prepared using CelLytic™ NuCLEAR™ Extraction Kit (Sigma) in accordance with the manufacturer's instructions. Protein lysates were separated with SDS-PAGE and transferred to the PVDF membranes (Millipore). After that, the membranes were cut into pieces according to the molecular weights of target proteins and incubated with the primary antibodies [AKT rabbit polyclonal antibody, 1:1000 (ProteinTech); CDK2 rabbit polyclonal antibody, 1:800 (ProteinTech); FOXO1 rabbit polyclonal antibody, 1:1000 (ProteinTech); phosphate FOXO1 rabbit polyclonal antibody, 1:800 (Abcam)] at 4°C for 12 h. The AlexaFluor 680/790-labeled goat anti-rabbit IgG antibody (1:10000, LI-COR Biosciences) was used as the secondary antibody. The blots were visualized by LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences).

PSpCas9(BB)-2A-GFP (pX458, Addgene, #48138) and pSpCas9(BB)-2A-Puro (pX459, Addgene, #48139) were purchased from Addgene (http://www.addgene.org). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was supplied by Aladdin Industrial Corporation (Shanghai, China). P-gp rabbit polyclonal antibody, PI3K mouse monoclonal antibody, mTOR mouse monoclonal antibody, Akt rabbit polyclonal antibody, p-PI3K rabbit polyclonal antibody, p-mTOR rabbit monoclonal antibody, and p-Akt mouse monoclonal antibody were products of Proteintech Group (Chicago, United States). 5-FU, DOX, paclitaxel, verapamil, and doxycycline were obtained from Sigma-Aldrich (Shanghai, China). All other reagents used in this study were of analytical grade and purchased from Sigma-Aldrich. Human breast cancer cell lines MCF-7 and MCF-7/DOX were purchased from the Type Culture Collection Committee of the Chinese Academy of Sciences (Shanghai, China). Fetal Bovine Serum (FBS), Roswell Park Memorial Institute 1,640 medium (RPMI 1640), Dulbecco’s Modified Eagle Medium (DMEM), and penicillin-streptomycin (10,000 U/mL penicillin and 10 mg/ml streptomycin) were obtained from Thermo Fisher Scientific (Waltham, United States).

LPS was obtained from Solarbio (China). Methanol, acetonitrile, and formic acid (chromatography pure) were obtained from Merck (Germany). PI3K mouse monoclonal antibody, p-PI3K rabbit monoclonal antibody, Akt rabbit polyclonal antibody, p-Akt mouse monoclonal antibody, Bcl-2 mouse monoclonal antibody, Bax mouse monoclonal antibody, GAPDH rabbit monoclonal antibody, HRP-conjugated AffiniPure goat anti-mouse IgG (H + L) and goat anti-rabbit IgG (H + L) were obtained from Proteintech Biotechnology (China).