Akt 1 2 3

Cell extracts were subjected to Western blot analysis as previously described [21 (link)]. The monoclonal antibody against the Myc-epitope (9B10) was from Millipore Technology. The expression vector of TNS2 was Myc-tagged and cloned into the pcDNA3.0 plasmid. To generate an antibody against p-Axl, the synthetic phospho-oligopeptide Asp-Gly-Leu-(phospho)Tyr-Ala-Leu-Met-Ser-Arg-Cys was used as an antigen for the generation of rabbit immune serum using a commercial service (GenTex Inc., California). The following antibodies were used: Axl (C-20), Erk sc-94, Santa Cruz), p-Erk (sc-7383, Santa Cruz), Akt 1/2/3 (#9272, Cell signaling) and p-Akt (#4051, Cell signaling), IRS-1 (#06–248,Millipore,), IRS-2 (#06–506, Upstate), TNS2 (SAB4200268, Sigma Aldrich), pTNS2-Y483 (ab138414, Abcam, Eugene), PDK1 (#21005, SAB signalway) and Glut4 (#2231S, Cell signaling).

The following antibodies were used at the indicated dilutions: PME‐1, Santa Cruz Biotechnology (Dallas, TX, USA) sc‐20086 (H‐226), Western blotting 1 : 1000; PME‐1, Santa Cruz Biotechnology sc‐25278 (B‐12), Immunohistochemistry 1 : 1000, Immunofluorescence 1 : 100; cleaved PARP‐1, Abcam (Cambridge, UK) ab32064 [E51], Western blotting 1 : 1000; GAPDH, HyTest 5G4‐6C5, Western blotting 1 : 5000; c‐MYC, Abcam ab32072 [Y69], Western blotting 1 : 1000; p‐Myc S62, Abcam ab78318, Western blotting 1 : 1000; AKT1/2/3, Cell Signaling Technology #9272, Western blotting 1 : 2000; p‐AKT S473, Cell Signaling Technology #4060, Western blotting 1 : 1000; Lamin‐A/C, Santa Cruz Biotechnology sc‐7292 (636), Immunofluorescence 1 : 250; Lamin‐A/C, Santa Cruz Biotechnology sc‐6215 (N‐18), Western blotting 1 : 1000; p‐Lamin‐A/C S392, Abcam ab58528, Western blotting 1 : 5000; EEA1, Santa Cruz Biotechnology sc‐137130 [G4], Immunofluorescence 1 : 100; p‐FAK Y397, Cell Signaling Technology #8556, Immunofluorescence 1 : 100; Histone H3K9me3, Cell Signaling Technology #13969 [D4W1U], Immunofluorescence 1 : 500; Histone H3K27me3, Cell Signaling Technology #9733 [C36B11], Immunofluorescence 1 : 500; PPP2R2A, Cell Signaling Technology #5689, Western blotting 1 : 1000.

Protein was extracted from homogenized tissue using the All-Prep DNA/RNA/protein mini kit, or by RIPA buffer. Proteins were resolved on 4–20% gradient gels (Bio-Rad, Sydney, Australia) and transferred onto Immobilon-P PVDF membrane (Millipore, Billerica, MA). Membranes were blocked with 5% skim milk or bovine serum albumin, followed by incubation with primary antibodies; β-actin (15 min, 1:30000, Sigma); insulin receptor-β (overnight, 1:2000, Santa Cruz Biotechnology); Akt1/2/3 (overnight, 1:2000, Cell Signaling) and phosphorylated Akt1/2/3 (p-Akt, overnight, 1:1000, Cell Signaling). Horseradish peroxidase-conjugated secondary antibodies (Dako) were used and signal detected by chemiluminescence (ECL^TM, Western blotting detection reagent, GE Healthcare, Chalfont St Giles, Buckinghamshire, UK).