Rabbit anti human phospho akt

His-tag recombinant human kallistatin was expressed in Pichia pastoris strain GS115 and purified by a series of chromatographic steps, mainly Phenyl Superose and Heparin Sepharose FF chromatography [24 (link), 25 ]. Rabbit anti-human integrin β3, rabbit anti-human phospho-Integrin β3, and mouse anti-human integrin β3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-human AKT, rabbit anti-human phospho-AKT, rabbit anti-human Erk1/2, rabbit anti-human phospho-Erk1/2, rabbit anti-human FAK, rabbit anti-human phospho-FAK, rabbit anti-human Src and rabbit anti-human phospho-Src were obtained from Cell Signaling Technology (Boston, MA). 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide was purchased from Sigma. Click-iT EdU Alexa Fluor 594 Imaging Kit (including Hoechst 33,342 and Apollo reaction cocktail) and Lipofectamine™ 2000 were from Life Technologies (Gaithersburg, MD). PVDF membranes and the Immobilon ECL detection system were purchased from Millipore Co (Billerica, MA). Integrin β3 and negative control siRNA were purchased from GenePharma (Shanghai, China).

UM-HMC cell lines were plated, serum starved overnight, treated with tocilizumab or Stattic (STAT3 inhibitor V, Calbiochem, San Diego, CA, USA) at the indicated concentrations, and exposed to 20 ng/ml rhIL-6 (R&D Systems, Minneapolis, MN, USA). NP-40 lysis buffer was used to prepare whole cell lysates that were resolved using PAGE. Membranes were incubated with the following primary antibodies for 1 hour at room temperature or overnight at 4°C: mouse anti-human phospho-STAT3, rabbit anti-human STAT3, rabbit anti-human phospho-ERK1/2, rabbit anti-human ERK1/2, rabbit anti-human phospho-AKT, rabbit anti-human AKT, rabbit anti-human Bcl-x_L, rabbit anti-human gp130 (Cell Signaling, Beverly, MA, USA), rabbit anti-human IL-6Rα, rabbit anti-human VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), hamster anti-human Bcl-2 (BD Pharmingen, Franklin Lakes, NJ, USA); or mouse anti-GAPDH (Chemicon, Billerica, MA, USA).

Western blotting was performed as reported previously [46 (link)]. The antibodies used were as follows: rabbit anti-human NECTIN4 (1:1000, Abcam; ab192033), rabbit anti-human Akt (1:1000, #9272), rabbit anti-human phospho-Akt (1:2000, #4060), rabbit anti-human ERK (1:1000, #9102), rabbit anti-human phospho-ERK (1:2000, #4370), rabbit anti-human MEK (1:1000, #9122), rabbit anti-human phospho-MEK (1:1000, #9154), rabbit anti-human EGFR (1:1000, #4267), rabbit anti-human β-actin (1:2000, #4970), and goat anti-rabbit IgG horseradish peroxidase-linked secondary antibody (1:10,000, #7074) (all from Cell Signaling Technologies, Danvers, MA, USA). Immunological bands were visualized with SuperSignal^TM West Pico Chemiluminescence Substrate (Thermo Fisher Scientific; 34580) and captured with the ChemiDoc^TM XRS Plus System (Bio-Rad Laboratories; 1708265J1PC). The signals of bands were measured with Image Lab Software (Bio-Rad Laboratories).