Rabbit anti mouse akt

Anesthetized mice were injected intradermally with 10 µl PBS in one ear and with 10 µl PBS containing 50 ng VEGF164 in the other ear. After 20 min, mice were culled, and the ear tissue surrounding the injection site was used for immunoblotting. These ear biopsies, as well as dermis and liver tissue from tamoxifen-treated mice and their littermate controls, was homogenized with a pestle, lysed in Laemmli sample buffer, and sonicated. Cells were lysed in 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.1% NP-40, 250 mM NaCl in the presence of protease inhibitor cocktail 2 and phosphatase inhibitor cocktail (Sigma-Aldrich). Denatured proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane (Whatman) for immunoblotting with the following primary antibodies: rabbit anti–human pVEGFR2-Y1175, rabbit anti–human pVEGFR2-Y951, rabbit anti–human VEGFR2, rabbit anti-SRC, rabbit anti–human pSRC-Y4169, rabbit anti–human pERK1/2-T202/Y204, rabbit anti–rat ERK1/2, rabbit anti–human pCRKL-Y207, rabbit anti–mouse AKT, rabbit anti-NRP1 (Cell Signaling Technology), rabbit anti–human GAPDH (Abcam), rabbit anti–human CRKL, and goat anti–human CDH5 (Santa Cruz Biotechnology, Inc.).

Everolimus, BGT226 AND BEZ235 were kindly provided by Novartis (Basel, Switzerland). Vincristine sulfate was purchased from Millennium Pharmaceuticals (Cambridge, MA), Doxorubicin from Pfizer (Melrose Park, NSW, Australia). Ionizing radiation was delivered using an X-ray irradiator (XRAD320, Precision X-Ray, Inc. East Haven, CT) at a dose rate of 0.91 Gy/minute. The following antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA): rabbit anti-phospho-4E-BP1, rabbit anti-4E-BP1, rabbit anti-phospho-S6RP, mouse anti-S6RP, rabbit anti-phospho-AKT (Ser⁴⁷⁵), rabbit anti-phospho-AKT (Thr³⁰⁸), rabbit anti-mouse AKT and rabbit anti-LC3B. Rabbit anti-cleaved caspase 3 was purchased from BD Pharmingen, (San Diego, CA, USA).

To evaluate the effect of MIH on (p)-Akt, (p)-AMPK, and OXPHOS complex protein expression in skeletal muscle, we performed a Western Blot analysis on protein lysates derived from the biopsies. Proteins were lysed, as described previously [25 (link)], and were subsequently homogenized. Next, a BCA assay was performed to determine protein lysate concentrations. Equal amounts of protein were loaded ([p]AMPK and [p]Akt 25 μg, OXPHOS 7.5 μg) and separated on the different gels. Primary antibodies included mouse monoclonal antibodies cocktail directed against human OXPHOS (dilution: 1:10.000; no. ab110411/no. MS601, Abcam/MitoSciences), rabbit anti-human AMPKα (1:1000, no. 2603, Cell Signaling), rabbit anti-human p-AMPKα^Thr172 (1:1000, no. 2535, Cell Signaling), rabbit anti-mouse Akt (1:1000, no. 9272, Cell signaling), and rabbit-anti mouse p-Akt^Ser473 (1:1000, no. 9271, Cell Signaling). Secondary antibodies were donkey anti-mouse conjugated with IRDYe800 (OXPHOS), swine anti-rabbit HRP (AMPKα, p-Akt, and Akt), and goat anti-rabbit HRP (p-AMPKα^Thr172). Visualization and analysis were performed using a CLx Odyssey Near Infrared Imager (OXPHOS) or ChemiDoc XRS system (AMPK, Akt) and Quantity One software. Individual intensities were quantified and are expressed in arbitrary units or derived ratios.