Total 4e bp1

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.

MV4-11 cells (5×10⁶ cells) were treated for 1 hour with AZD-8055 at concentrations ranging from 25–1000 nM and with vehicle DMSO. Cell pellets were lysed in 150 μl Bicine Chaps lysis buffer (containing protease and phosphatase inhibitor cocktail made as per Protein Simple specifications). Protein concentrations were determined by Bio-Rad protein assay. Proteins were separated using SDS-polyacrylamide gels, transferred to polyvinylidene diflouride membranes (EMD Millipore) and blocked in 5% non-fat dry milk. Primary antibody incubations were performed overnight at 4°C, followed by incubation in secondary horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibody at room temperature for 1 hour. Primary antibodies used were total 4EBP1 (Cell Signal Cat #9644 s), phospho-serine 65 4EBP1(Cell Signal Cat #9451 s), phospho-threonine 37/46 4EBP1 (Cell Signal Cat #2855), total Akt 1/2/3 (Santa Cruz Cat #sc-8312), β-2 microglobulin (Abcam Cat #ab75853) and β-actin (Sigma Cat #A5441). These were used at a concentration of 1:1000 except for β-actin (conc. 1:10,000) and secondary antibodies (Anti-mouse Cell Signal Cat #7076S, Anti-rabbit Cell Signal Cat #7074S) were used at a concentration of 1:2000.