Rabbit anti phospho akt ser473

Western blot analysis was performed as previously described, with slight modifications [11 (link)]. Briefly, HUVEC lysates were lysed using RIPA buffer supplemented with protease inhibitor (Cell Signaling 5871S). Proteins were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel, transferred to a PVDF membrane (GE Healthcare, RPN303F), and blocked in 5% bovine serum albumin (BSA) in PBS with 1% tween-20 (Sigma P2287) for 1h at RT. Primary antibodies used were: rabbit anti-phospho-Smad1/5 (1:1000, Cell Signaling 9516), rabbit anti-Akt (1:1000, Cell Signaling 9272), rabbit anti-phospho-Akt (Ser473) (1:1000, Cell Signaling 4060), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (1:1000, Cell Signaling 4370), rabbit anti-ERK 1/2 (1:1000, Cell Signaling 4695), mouse anti-HIF1α (1:500, Novus biologicals NB100-105), mouse anti-p53 (1:1000, Abcam ab1101) and rabbit anti-p53 (1:500, Abcam ab131442). Membranes were incubated with primary antibodies diluted in 1% BSA overnight at 4°C. Signal was detected with horseradish peroxidase (HRP) anti-rabbit (1:5000, Invitrogen G-21234) or HRP anti-mouse (1:30,000, Invitrogen 81–6720), and imaged via Clarity Western ECL Substrate (Bio-Rad 170–5061). Full original blots are shown (S6 Fig).

The muscle supernatants were subjected to Western blotting as described previously [31 (link)]. Phosphorylation of mTOR at Ser2448 was detected using rabbit anti-phospho-mTOR (Ser2448) (Cell Signaling Technology, Danvers, MA, USA) and expressed as the ratio of total mTOR expression, determined using anti-mTOR (Cell Signaling Technology, Danvers, MA, USA). Phosphorylation of Akt at (Ser473) was detected using rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA) and expressed as a ratio of total Akt expression, determined using anti-Akt (Cell Signaling Technology, Danvers, MA, USA).

Cell lines Huh-7 and PLC/PRF/5 were bought from Cell Bank of Chinese Academy of Sciences. Cells were maintained in DMEM media (#12800017; GIBCO) supplemented with 10% fetal bovine serum (#10437-028; GIBCO) and 1.5 g/L of NaHCO₃. The protein samples were blotted with following antibodies: rabbit anti-Cyclin D1 (#2978 S; Cell Signaling Technology) 1:1000, mouse anti-CDK6 (#3136 S; Cell Signaling Technology) 1:1000, rabbit anti-Phospho-Akt(Thr308) (#9275 S; Cell Signaling Technology) 1:600, rabbit anti-Phospho-Akt(Ser473) (#9271 S; Cell Signaling Technology) 1:600, rabbit anti-c-Jun (60A8) (#9165 S; Cell Signaling Technology) 1:1000, rabbit anti-B-Raf (#sc-166; Santa Cruz Biotechnology) 1;400, rabbit anti-Cleaved Notch1 (Val1744) (#4147 S; Cell Signaling technology)1:300, rabbit anti-β-Catenin (#8480 S; Cell Signaling technology) 1:1000, rabbit anti-LC3B (#ab48394; abcam) 1:1000, mouse anti-GSTP1 (#3369 S; Cell Signaling technology) 1:1000, mouse anti-Actin (#HC201; TransGen Biotech) 1:3000. BALB/c nude mice were housed in laminar flow cabinets with free access to food and water in Laboratory Animal Center of Fujian Medical University. All of animal experiments were performed in accordance with the animal protocols and regulations approved by FJMU Experimental Animal Ethics Committee of Fujian Medical University.

For immunohistochemistry, the sections were incubated at 4°C overnight with 1∶100 dilutions of primary antibodies, including rabbit anti-active caspase 3 (BD Biosciences, San Jose, CA, USA), mouse anti-PCNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-endothelin 1 (GeneTex Inc., Hsinchu, Taiwan), rabbit anti-phospho-AKT (Ser473) (Cell Signaling Technology, Danvers, MA, USA), and anti-AKT (Cell Signaling Technology, Danvers, MA, USA). After washing with PBS, the sections were incubated with a 1∶100 dilution of the secondary antibody, either goat anti-rabbit IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or goat anti-mouse IgG (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), at room temperature. The protocol of immunohistochemistry was described previously [32] (link).

The following antibodies were obtained from commercial sources: rabbit anti-green fluorescent protein (GFP; catalog no. 598, Medical and Biological Laboratories, Woburn, MA, USA), rabbit anti-S6 ribosomal protein (catalog no. 2217, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-S6 (Ser-240/244, catalog no. 2215, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser-473, catalog no. 4060, Cell Signaling Technology), mouse anti-Akt (catalog no. 2920, Cell Signaling Technology), mouse anti-β-galactosidase (catalog no. Z3781, Promega, Madison, WI, USA), rabbit anti-β-galactosidase (catalog no. 559762, MP Biomedicals, Santa Ana, CA, USA), mouse anti-monoubiquitinylated and polyubiquitinylated conjugates (clone FK; catalog no. BML-PW8810, Enzo Life Sciences, Farmingdale, NY, USA), and mouse anti-tubulin (catalog no. T5168, Sigma-Aldrich, St. Louis, MO, USA). Anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 647 were obtained from Invitrogen (Carlsbad, CA, USA). IRDye-conjugated secondary donkey anti-mouse IRDye 680RD antibody (catalog no. 926-68072) and donkey anti-rabbit IRDye 800CW antibody (catalog no. 926-32213) were obtained from LI-COR Biosciences (Lincoln, NE, USA). Insulin was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Immunoblotting was performed using the following antibodies: rabbit anti-Bmi-1 (#5856; Cell Signaling Technology, Inc., Beverly, MA, USA), rabbit anti-p16 (sc1207; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rat anti-p19Arf (sc32748; Santa Cruz Biotechnology, Inc.), rabbit anti-p15 (sc613; Santa Cruz Biotechnology, Inc.), rabbit anti-p18 (sc865; Santa Cruz Biotechnology, Inc.), rabbit anti-phospho-Rb (ser807/811) (#9308; Cell Signaling Technology, Inc.), rabbit anti-phospho-Akt (ser473) (#4060; Cell Signaling Technology, Inc.), rabbit anti-Akt (#4691; Cell Signaling Technology, Inc.), and mouse anti-β-actin (A5316; Sigma-Aldrich, St. Louis, MO, USA).