Mouse anti akt

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).

Chlorhexidine gluconate (CG) was purchased from Sigma-Aldrich (St. Louis, MO, USA). RAPA was purchased from Solarbio (Beijing, China). BEZ235 was obtained from Selleck (Houston, TX, USA). The mouse anti-fibronectin (FN), mouse anti-α-smooth muscle antigen (α-SMA), and mouse anti-collagen 1A1 (Col 1) monoclonal antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). The rabbit anti-mTOR, rabbit anti-phospho-mTORSer2448, mouse anti-phospho-AktSer437, mouse anti-Akt, mouse anti-phospho-p70S6KThr389, and rabbit anti-p70S6K monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The immunohistochemistry ultra-sensitive SP reagent kit was obtained from BioTNT (Shanghai, China).

Nitrocellulose membranes containing the transferred proteins were blocked in Intercept Blocking Buffer (LI-COR Bioscience) for 1-hour at room temperature. Membranes were probed with primary antibodies, including mouse anti-mTOR (1:1000; Cat# 4517), mouse anti-Akt (1:1000; Cat# 2920), mouse anti-GSK3β (1:1000; Cat# 9832), mouse anti-ERK (1:1000; Cat# 9107), rabbit anti-phospho mTOR (1:1000; Cat# 5536 S), rabbit anti-phospho Akt (1:1000; Cat# 4060 S), rabbit anti-phospho GSK3β (1:1000; Cat# 5558), rabbit anti-phospho ERK (1:1000; Cat# 4376) (all antibodies were purchased from Cell Signaling Technologies) diluted in Intercept Blocking Buffer + 0.2% Tween-20 and incubated overnight at 4 °C, before being exposed to secondary antibody (IRDye^® 680RD Donkey anti-Mouse IgG and IRDye^® 800CW Donkey anti-Rabbit IgG) (LI-COR Biosciences) for 1-hour at room temperature in the dark.
Membranes were scanned on the LI-COR Bioscience Odyssey CLx imaging system and imaged using LI-COR Image Studio software version 2.1.10. All densitometry analyses were performed using Image Studio Light version 5. The region of interest encircling each band was defined automatically. All bands at the correct molecular weight were analyzed as the signal for that target protein. Values for each protein were normalized to loading control β-tubulin (Abcam).

HS01 cells were seeded at 250,000 cells/well in 6-well CellBIND (Corning) plates, grown to ~80% confluency, then treated with CUDC907 for indicated times. Cells were extracted with 4% sodium dodecyl sulfate (SDS), 0.01% bromophenol blue, 10% glycerol and 100 mmol/L dithiothreitol. Antibodies were used from Cell Signaling Technology: rabbit anti-cleaved caspase 7 (1:500; #8438); rabbit anti-cleaved caspase 3 (1:1000, #9664); rabbit anti-acetylated lysine (1:1000; #9814); mouse anti-actin (1:30,000; #3700); rabbit anti-pFAK(Y397) (1:1000; #8556), mouse anti-FAK (1:500; #3285); rabbit anti-pERK (1:1000; #4370); mouse anti-ERK (1:500; #9107); rabbit anti-pAKT (T308) (1:1000; #29655); mouse anti-AKT (1:1000; #29205); rabbit anti-pAKT (S473) (1:1000; #4060); rabbit anti-YAP (1:1000; #14074); rabbit anti-pYAP (S127) (1:1000; #4911). Primary antibodies were diluted in 1:1 tris-buffered saline-0.1% Tween and Odyssey Blocking Buffer or 5% milk and incubated overnight at 4°C. Secondary antibodies were diluted in same solution and incubated for 45 minutes at room temperature. Western blots were imaged on the LI-COR Odyssey Imaging System (LI-COR Biosciences) or ChemiDoc (Bio-Rad) and quantified on ImageJ (NIH).

Cells were scraped in PBS, and then suspended in RIPA buffer (Sigma-Aldrich), with added protease and phosphatase inhibitors (EMD Millipore). They were then homogenized and centrifuged for 5 minutes at 800 g. The Western blot experiments were performed using the supernatant from this centrifugation. Protein samples were denatured at 100°C for 3 min. 20 μg of total protein samples were loaded into each well of SDS-PAGE 4-20% precast gels (Bio-Rad, Hercules, CA, USA) and underwent electrophoretic separation at 200 V for 50 minutes in running buffer. Proteins were then transferred to Hybond nitrocellulose membranes (Bio-Rad) by electro-blotting. The blocking phase was performed with 5% not-fat dry milk (Bio-Rad) in 0.1% PBS-Tween 20 (Sigma-Aldrich), followed by incubation with the primary antibodies diluted in the same solution. The following antibodies, all from Cell Signaling Technologies, were used: mouse anti-Akt 1:500; rabbit anti-pAkt Ser473 1:200; rabbit anti-pERK 1/2 1:500; mouse anti-ERK 1/2 1:200. After a rinse in PBS, membranes were incubated in PBS-Tween 20 0.1% containing suitable IRDye secondary antibodies. The antibodies, both from LI-COR Biosciences (Lincoln, NE, USA), were used at 1:7000 dilution: goat anti-rabbit IRDye 800; goat anti-mouse IRDye 680. After washing, the IR signal was detected with an Odyssey scanner (LI-COR Biosciences).

Cells were seeded at 4 × 10⁵ per well in six-well plates with appropriate growth medium and incubated at 37 °C, 5% CO₂ overnight, or treated with drugs for indicated period of time before harvesting. Cell lysates were prepared by cell lysis on ice with 1X RIPA buffer (Cell Signaling, #9806) containing protease inhibitor (Roche, #11836170001) and phosphatase inhibitor cocktail (Sigma, #P5726). Immunoblotting of individual protein bands was performed by incubating the PVDF membranes with the following primary antibodies (all purchased from Cell Signaling) diluted 1:1000 in 5% BSA/TBST: rabbit anti-phospho-ERK (#9101), mouse anti-ERK1/2 (#9107), rabbit anti-phospho-AKT (Ser473) (#4060), mouse anti-AKT (#2920), rabbit anti-phospho-FAK (Y397) (#8556), rabbit anti-FAK (#13009), and rabbit anti-GAPDH (#2118). Fluorescent Alexa 488-conjugated donkey anti-rabbit or anti-mouse antibodies (Life Technologies, A21206 or A21202) were used as secondary antibodies diluted 1:5000 in 5% BSA/TBST to visualize the protein bands on the Pharos Molecular Imager (BioRad). Intensities of individual protein bands from the scanned images were measured using ImageJ after subtracting background. Phosphoprotein levels were then normalized to either GAPDH or total level of the corresponding protein. Uncropped images of immunoblots are shown in Supplementary Figures 7 to 11.