Western Blot Analysis of AKT Signaling

Western analyses were performed as described25 (link). For each experiment, lysates from a cell line grown with or without serum were run on the same gel and all gels were run at the same time. Two gels were transferred to a single membrane. If more than one membrane was used for an experiment, a single solution for each primary and secondary antibody pair was made and divided between the two membranes that were subsequently hybridized in separate trays. Antibodies used included pAKT(S473) (product #4060), pAKT(T308) (product #2965); pan-AKT (product #2920 and 4691), p PRAS40 (product #2997), PRAS40 (product # 2610), pS6(S235) (product #2211), pS6(S240) (product #5364) S6 (product #2317 and 2217), pFOXO1/FOXO3a (product #9464), FOXO3a (product #2497), p GSK3α/β (product #9331), GSK3α/β (product #5676), pBAD (product #4366) and BAD (product #9239) from Cell Signaling Technologies (Danvers, MA, USA) or β-actin (product #A5441) from Sigma-Aldrich (St. Louis, MO, USA). Membranes were scanned and intensities calculated using a semi-automated infrared imaging system (Odyssey scanner and Image Studio Lite software, LiCor BioSciences Inc., Lincoln, NE, USA). Histograms show ratios of infrared signal intensities of indicated antibodies. For experiments using two membranes, histograms of infrared signal ratios graphed by membrane are shown in Supplementary Fig. 8.

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Lindhurst M.J., Yourick M.R., Yu Y., Savage R.E., Ferrari D, & Biesecker L.G. (2015). Repression of AKT signaling by ARQ 092 in cells and tissues from patients with Proteus syndrome. Scientific Reports, 5, 17162.

Publication 2015

pAKT(S473) pAKT(T308) pan-AKT pPRAS40 PRAS40 pFOXO1/FOXO3a FOXO3a pGSK3α/β GSK3α/β pBAD β-actin

Actin Antibodies Antibody Cell line Gels Gsk3α Membrane Serum

Corresponding Organization :

Other organizations : National Human Genome Research Institute, National Institutes of Health, Arqule (United States)

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Variable analysis

independent variables

Serum treatment (with or without serum)

dependent variables

Phosphorylation levels of AKT (S473 and T308)
Phosphorylation levels of PRAS40
Phosphorylation levels of S6 (S235 and S240)
Phosphorylation levels of FOXO1/FOXO3a
Phosphorylation levels of GSK3α/β
Phosphorylation levels of BAD

control variables

Gel run conditions (all gels were run at the same time)
Membrane transfer conditions (two gels were transferred to a single membrane)

controls

Positive control: Antibodies for total AKT, PRAS40, S6, FOXO3a, GSK3α/β, and BAD
Negative control: β-actin antibody

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