P akt ser473 rabbit mab

Trastuzumab (Herceptin) was provided by Genentech and used directly. Meo-PEG_5k-Dlink_m-PLGA_11k copolymer and G0-C14 were synthesized using our published methods54 (link), 55 , 56 (link). PTEN mRNA, EGFP mRNA, and fluorescent dye Cy5-labeled mRNA (Cy5-EGFP mRNA and Cy5-PTEN mRNA) were purchased from APExBIO (Houston, TX, USA). PTEN rabbit mAb (#ab32199), HER2 rabbit mAb (ab214275), Ki67 rabbit mAb (#ab92742), and GAPDH rabbit mAb (#ab181602) were purchased from Abcam. Anti-rabbit IgG horseradish peroxidase (HRP)-linked secondary mAb (#7074), Akt (Pan) (C67E7) rabbit mAb (#4691), p-Akt (Ser473) rabbit mAb (#4060), and cleaved caspase-3 rabbit mAb (#9664) were purchased from Cell Signaling Technology (CST). Alexa Fluor 647-conjugated secondary antibody (#A-11011) and Click-iT™ TUNEL Colorimetric IHC Detection Kit (#C10625) were obtained from ThermoFisher. Fetal bovine serum (FBS), penicillin-streptomycin, Dulbecco's modified Eagle medium (DMEM), and trypsin were obtained from Invitrogen Corp. All solvents and reagents are of analytical grade and used directly.

Cells were washed in cold PBS and lysed in NP-40 lysis buffer (Beyotime, P0013F). Lysates were incubated on ice for 30 min and cleared by centrifugation at 12,000g for 10 min. Protein concentration was quantified using a BCA Protein Assay Kit (Beyotime, P0012). Total protein was mixed with sample buffer and denatured at 100°C for 10 min. Sample was separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, IPVH00010). Membranes were blocked with 5% (w/v) nonfat dry milk and incubated with primary antibodies β-actin rabbit monoclonal antibody (mAb; Abmart, P30002), PI3Kγ rabbit mAb (Zenbio, R25369), p-AKT (Ser⁴⁷³) rabbit mAb (Cell Signaling, #9271), GPX4 rabbit mAb (ABclonal, A1933), ACSL3 rabbit mAb (Zenbio, 161226), and ACSL4 rabbit mAb (Zenbio, R24265), overnight at 4°C followed by washing in 0.1% Tween/PBS. Membranes were incubated with corresponding secondary antibodies at 25°C for 1 hour and then washed three times before signal detection.