Total akt

U87 cells were washed with ice-cold PBS before lysis in RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0). Cells were scraped off from six-well plates and agitated at 4°C for 10 min. After centrifugation at 12,000 rpm at 4°C for 20 min, supernatants were mixed at 1∶1 ratio with 2× Laemmli buffer. Protein lysates were heated at 95°C for 5 min, separated by 10% Mini-PROTEAN gels (Bio-Rad), transferred onto 0.45 µm nitrocellulose membranes, and probed with antibodies against p-STAT1 (Y701), total STAT1, NF-κB p65, p-Akt, total Akt, p-mTOR, total mTOR, PKR (Cell Signaling), VSV, GFP, NF-YA, NF-YB, NF-YC, p-p70 S6 kinase, total p70 S6 kinase, PI3K, p21, Rb, caspase-3, caspase-9, XIAP, survivin, β-tubulin (Santa Cruz), acetylated H3 (Millipore), and total histone H3 (Abcam). Secondary incubation was performed with anti-goat, anti-rabbit, or anti-mouse IgG HRP-linked antibodies (Cell Signaling). Proteins were visualized using HyGlo chemiluminescent HRP antibody detection reagent (Denville Scientific Inc.) and a Fuji LAS-3000 cooled CCD camera (FUJIFILM).

Cells were lysed on ice for 30 min with RIPA lysis buffer, which contains 50 mM Tris^_HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors (10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 10 mg/ml aprotinin). For western blotting, 25 μg of protein sample was resolved on 12.5% SDS-PAGE and electrotransferred onto nitrocellulose membranes (Whatman). The primary antibodies used were as follows: phospho-Akt, total Akt, phospo-p44/p42 ERK, total p44/p42 ERK, phospho-p38, total-p38, phospo-JNK, and total-JNK were purchased from Cell Signaling Technology (1:1000 dilution ratio). Anti-NFATC1 antibody was purchased from Santa Cruz (1:500 dilution ratio). β-actin or GAPDH was used as loading control. Horseradish peroxidase-conjugated secondary antibodies were used at a 1:5000 dilution. The antigen–antibody complexes were visualized using the enhanced chemiluminescence detection system (Millipore) following the manufacturer’s instructions. Immunoreactive bands were quantitatively analyzed in triplicate by normalizing the band intensities to their respective controls on scanned films using ImageJ software.

Tissue samples were collected and flash frozen. After 24 h, protein was extracted and immunoblotting performed as previously described.^{43 (link)} The membranes were blocked with 5% non-fat dry milk for 1 h and then probed with primary antibodies against phospho RPS6 (Ser235/236, 4858, Cell Signaling Technology), phospho AKT (Ser473, 4060, Cell Signaling Technology), phospho 4EBP1 (Thr37/46, 2855, Cell Signaling Technology), total RPS6 (2217, Cell Signaling Technology), total AKT (4691, Cell Signaling Technology) or total 4EBP1(9644, Cell Signaling Technology) in bovine serum albumin at 1:1000. Anti-GAPDH antibody (8884, Cell Signaling Technology) was used as a loading control at a ratio of 1:5000.

Human thrombin, PGE1, dimethyl sulfoxide (DMSO), ADP, fibrinogen, TXA₂ analog U46619, ferric chloride (FeCl₃), and all the reagents were purchased from Sigma (St. Louis, MO, United States). D-Phe-Pro-Arg-chloromethyl ketone (PPACK) was purchased from EMD Millipore (Billerica, MA, United States). Collagen-related peptide (CRP) was obtained from Dr. Richard Farndale (Department of Biochemistry, University of Cambridge, United Kingdom). Phycoerythrin (PE)-conjugated isotype control IgGs, rat monoclonal antibodies against mouse P-selectin, and activated αIIbβ3 (JON/A) were obtained from Emfret Analytics (Eibelstadt, Germany). Antibodies against phospho-PLCγ2 at Tyr759, phospho-PLCβ3 at Ser1105, phospho-Akt at Ser473, Total Akt, Total PLCγ2, Total PLCβ3, and actin were obtained from Cell Signaling (Danvers, MA, United States). Calcium dye (FLIPR Calcium Assay kit) was obtained from Molecular Devices (Sunnyvale, CA, United States).

Blotting was performed as per our previous reports32 (link)33 (link). Briefly, total protein of lysates was extracted and equal amount of protein was loaded onto 10% sodium dodecyl sulphate polyacrylamide gel for electrophoresis. Gels were subsequently transferred to nitrocellulose membrane. The membranes were blockaded for 1 h with 5% non-fat milk. Primary antibodies against VHL (Abcam), HIF1α (Abcam), HIF2α (Abcam), SETD2 (Abnova), H3 (Abcam), H3K36me2 (Lys36, Cell Signaling), H3K36me3 (Lys36, Cell Signaling), PI3 Kinase p110β (Cell Signaling), pS6 (Ser235/236, Cell Signaling), total S6 (Cell Signaling), pAkt (Ser473, Cell Signaling) and total Akt (Cell Signaling) were then added and membranes were kept incubating at 4°C overnight. Corresponding secondary antibodies were applied followed by electrochemiluminescence (ECL) processing.

Proteins were isolated from the organic phase of the TRIzol (Thermo Fisher Scientific) tissue lysate, which was a side product from the RNA isolation procedure. Proteins were precipitated according to the manufacturer’s instructions. Equivalent amount of proteins were resolved by 10% SDS-PAGE and immunoblotting using antibodies directed against P-Akt (Thr308 for human Akt corresponding to Thr307 for zebrafish Akt, 244 F9; cat. no. 4056, Cell Signaling), total Akt (cat. no. 9272, Cell Signaling) and beta-Actin (cat. no. ab8227, abcam). Protein levels of P-Akt and total Akt were analyzed using ImageJ software (National Institutes of Health) and the ratio was calculated.