P pras40 thr246

Boiled whole cell lysates were run on a precast Tris-acetate (Thermo Fisher Scientific) or Mini-PROTEAN TGX protein gel (Bio-Rad) and then transferred to a nitrocellulose membrane (Bio-Rad). The membrane was incubated overnight at 4 °C with primary antibodies against Cas9 (1:1,000; Active Motif 61577), Pten (1:1,000; Cell Signaling Technology 9188), ERG (1:1,000; Abcam ab92513), p53 (1:1,000; Cell Signaling Technology 2524), Apc (1:1,000; Millipore MABC202), AKT1 (1:2,000, Cell Signaling Technology 4060L), AKT2 (1:1,000, Cell Signaling Technology 5239S), pPRAS40-Thr246 (1:1,000, Cell Signaling Technology 2997S), Actin-HRP (horseradish peroxidase) (1:10,000; Abcam ab49900), Cyclophilin B (1:10000; Cell Signaling Technology 43603S) and Vinculin (1:10,000; Cell Signaling Technology 13901). Signal was visualized with secondary HRP-conjugated antibodies and chemiluminescent detection.

Cells and lungs from dissected animals of all genotypes and cell lines were homogenized and lysed in 50 nM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1 mM sodium orthovanadate (Na₃VO₄), 10 mM NaF, and protease inhibitor cocktail (Roche) and cleared by centrifugation; concentrations were determined by protein assay (Bio-Rad Laboratories), and samples were combined with SDS sample loading buffer followed by brief sonication and centrifugation. The supernatant was collected for Western blotting. Western blotting was done using the following polyclonal antibodies from Cell Signaling Technology: Myc-tag (#2272), p-Akt Ser⁴⁷³ (#4051), p-S6 ribosomal protein Ser^235/236 (#4856), and p-PRAS40 Thr²⁴⁶ (#2997), as published. Other polyclonal antibodies used were IPO11 (AP9661b; Thermo Fisher Scientific), UBE2E1, UBE2E3, NEDD4-1 (07-049; EMD Millipore), Ndfip1 (HPA009682; Sigma-Aldrich), and Ub (P4D1; Santa Cruz Biotechnology, Inc.). mAbs used were HA-tag (12CA5; homemade), β-actin (A5441; Sigma-Aldrich), γ-tubulin (T6199; Sigma-Aldrich), GFP (JL-8; Takara Bio Inc.), and c-Myc (ab32072; Abcam). Western blots were developed using the enhanced chemiluminescence detection reagent (EMD Millipore) or the Odyssey Infrared Imaging System (LI-COR Biosciences). Quantification was done using the LI-COR system or densitometry using ImageJ 1.38X software (National Institutes of Health).

The monoclonal antibodies against ADCY5, pmTOR(S2448), mTOR, pPRAS40 (Thr246), PRAS40, FoxO3a, LC3B, GSK-3β (S9P), IR, PCDNA and pJNK, senescence β-gal staining kit, and SimpleChIP chromatin IP kit were obtained from Cell Signaling (Beverly, MA, USA). Polyclonal antibodies against MKP7, RGS2, pEGFR, EGFR, pERK, ERK2, pMEK1, MEK1, IRS1, Cav2, cyclin D1, and c-myc were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The monoclonal antibodies against HIF1α, pAKT, and β-actin were obtained from Abcam (San Francisco, CA, USA). Horseradish peroxidase-conjugated goat anti-mouse IgG and horseradish peroxidase-conjugated goat anti-rabbit IgG were obtained from Bio-Rad (Hercules, CA, USA). Immunoblotting was performed using the ECL western blot detection kit (enhanced chemiluminescence; Amersham Biosciences, Amersham, UK). miRNA RT and PCR kits were obtained from Qiagen (Valencia, CA, USA). cAMP assay kit was obtained from R&D (Minneapolis, MN, USA).