Rabbit monoclonal anti p akt

In order to further investigate the possible mechanisms of NSCs’ effect on tumor cell behavior changes, expression of mutant p53, caspase-3 and the phosphorylation status of ERK1/2, AKT were detected by Western blot assay. Rabbit monoclonal anti-mutant p53 (1:1,000, abcam, USA), rabbit monoclonal anti-GFAP (1:2,000, Millopore, USA), rabbit polyclonal anti-caspase 3 (1:1,000, Cell Signaling, USA), rabbit polyclonal anti-AKT (1:1,000, Cell Signaling, USA), rabbit monoclonal anti-p-AKT (1:1,000, Cell Signaling, USA), rabbit monoclonal anti-ERK1/2 (1: 1,000, Cell Signaling USA), rabbit monoclonal anti-p-ERK 1/2 (1: 1,000, Cell Signaling USA) were used.

Specific reagents used in this study were actinomycin D, G418, hygromycin B, puromycin, sodium butyrate, TriPure Isolation Reagent solution, and valproic acid from Millipore Sigma, doxycycline and NuPAGE^™ LDS Sample Buffer (4×) from Thermo Fisher Scientific, radioimmunoprecipitation assay buffer (RIPA) from Boston Bioproducts (Ashland, MA), and LipoD293^™ In Vitro DNA Transfection Reagent from SignaGen Laboratories (Frederick, MD). Following antibodies were used in this report: affinity-purified rabbit polyclonal anti-ORF57 N-terminal (in house), mouse monoclonal anti-ORF57 N-terminal, and affinity-purified rabbit polyclonal anti-ORF57 C-terminal antibodies were from Rockland Immunochemicals. Rabbit polyclonal anti-AKT, rabbit monoclonal anti-p-AKT, rabbit monoclonal anti-c-FOS (9F6), mouse monoclonal anti-GAPDH, and rabbit polyclonal anti-c-JUN antibodies were purchased from Cell Signaling Technology. Rabbit polyclonal anti-RTA was a gift of Dr. Yoshi Izumiya (UC Davis). Mouse monoclonal anti-myc (9E10) was a gift of Dr. Xuefeng Liu (the Ohio State University). Mouse polyclonal anti-AEN was from Millipore Sigma, rabbit polyclonal anti-RGS2 from Abcam, rabbit polyclonal anti-ZFC3H1(MTR4) antibody from Novus biologicals, and rabbit IgG isotype, mouse IgG isotype were from Thermo Fisher Scientific.

Human normal bronchial epithelial cells (HBE), human monocytic cells (U937), and human lung cancer cells (A549) were obtained from Shanghai Chinese Academy of Science. Cells were cultured into the RPMI 1640 complete medium (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France) and 1% penicillin‐streptomycin antibiotics (PS; Genom, Hangzhou, China) in 5% CO₂ at 37°C. Hypoxic culture was performed in the three‐phase incubator at 37°C, 1% O₂, 5% CO₂, and 90% relative humidity. Human recombinant OPN (R&D, Shanghai, China), PI3K/Akt specific inhibitor SHBM1009 (Biovision, San Francisco, USA), Erk1/2 specific inhibitor PD98059 (Biovision, San Francisco, USA), mouse monoclonal anti‐vimentin antibody (Abcam, Hong Kong, China), and rabbit polyclonal to OPN (Abcam, Hong Kong, China) were used in this study. Rabbit monoclonal anti‐p‐Akt, p‐Erk1/2, total‐Akt, total‐Erk1/2, and E‐cadherin were purchased from Cell Signaling Technology Company (Danvers, MA). Cell‐IQ live cell imaging platform was manufactured by Chipmantech (Tampere, Finland).