Akt1 2 3 sc 8312

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Akt1/2/3 (sc-8312) is a mouse monoclonal antibody that recognizes the Akt1, Akt2, and Akt3 proteins. Akt is a serine/threonine protein kinase that plays a key role in multiple cellular processes, including cell proliferation, survival, and metabolism.

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Lab products found in correlation

β actin, by Merck Group (2 mentions) P akt ser473, by Cell Signaling Technology (1 mentions) Bio 1d software, by Avantor (1 mentions) Ab14955, by Abcam (1 mentions) Ab45899, by Abcam (1 mentions) Hoechst, by Merck Group (1 mentions) Hrp linked antibodies, by Santa Cruz Biotechnology (1 mentions) Novex ecl chemi substrate, by Thermo Fisher Scientific (1 mentions) Pgc 1α nbp1 04676, by Novus Biologicals (1 mentions) Phospho akt ser473, by Cell Signaling Technology (1 mentions) Hrp conjugated secondary antibody, by Rockland Immunochemicals (1 mentions)

3 protocols using akt1 2 3 sc 8312

Comprehensive Gene Expression Analysis

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RNA isolation, cDNA synthesis and qPCR were performed at day 8 of differentiation (or as stated in the figure legends) as previously described [6] (link). Control reactions for contaminating DNA were performed routinely and relative expression calculated using the Pfaffl method [29] (link). The geometric mean of three stable reference genes (Nono, Ywhaz and Actb or as stated in the figure legend) were obtained from five commonly used sequences and used for normalisation. Primer sequences available on request, some of which were obtained from PrimerBank [30] (link).
SDS-PAGE was performed and transferred to nitrocellulose membranes as described previously [31] (link). Antibodies against p-eIF2α (#9721), eIF2 α (#5324S), p-p38 MAPK (#9211), p38 MAPK (#8690S), Beclin1 (#3495), LC3B (#3868), p-Akt Ser473 (#9271) were from Cell Signalling, SH-PTP2 (sc-280) and Akt1/2/3 (sc-8312) from Santa Cruz. All antibodies were detected with goat anti-rabbit HRP secondary antibody (#28177) from Anaspec. Proteins were visualized using enhanced chemiluminescence (ECL) and quantified by densitometry scanning using the Fusion imaging system and Bio-1D software (Peqlab).

Mcilroy G.D., Tammireddy S.R., Maskrey B.H., Grant L., Doherty M.K., Watson D.G., Delibegović M., Whitfield P.D, & Mody N. (2016). Fenretinide mediated retinoic acid receptor signalling and inhibition of ceramide biosynthesis regulates adipogenesis, lipid accumulation, mitochondrial function and nutrient stress signalling in adipocytes and adipose tissue. Biochemical Pharmacology, 100, 86-97.

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Immunoblot and Immunofluorescence Assays

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Immunoblot and immunofluorescence assays were performed following standard procedures, as previously described [40 (link)]. Primary antibodies were: phospho Histone H3 Ser10 (ab14955, Abcam), TOMM20 (11802-1-AP, Proteintech), PGC1-α (NBP1-04676, Novus Biologicals), phospho p38MAPK Thr180/Tyr182 (9211, Cell Signaling), p38MAPK (sc-7972, Santa Cruz Biotechnology) AKT1/2/3 (sc-8312, Santa Cruz Biotechnology), phospho AKT Ser 473 (9271, Cell Signaling), DMPK (sc-134319, Santa Cruz Biotechnology), MBNL1 (ab45899, Abcam) and β-actin (AC-15, Sigma-Aldrich). For western blot detection of primary antibodies, we used HRP-linked antibodies (Santa Cruz Biotechnology) and the detection was performed by chemiluminescence using Novex ECL Chemi Substrate (Thermo Fisher). For immunofluorescence, nuclear DNA was stained with Hoechst (33342, Sigma-Aldrich).

García-Puga M., Saenz-Antoñanzas A., Fernández-Torrón R., de Munain A.L, & Matheu A. (2020). Myotonic Dystrophy type 1 cells display impaired metabolism and mitochondrial dysfunction that are reversed by metformin. Aging (Albany NY), 12(7), 6260-6275.

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Western Blot Analysis of Protein Expression

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Total cell lysates were prepared with 1×SDS lysis buffer with 100mM Dithiothreitol and 2µg/ml protease inhibitors containing 0.1mM leupeptin, aprotinin, and pepstatin. After electrophoresis, proteins were transferred to nitrocellulose membrane, blocked with 5% nonfat milk, and probed overnight with primary antibodies against Gli1 (1:200; Abcam, MA, USA), β-actin (1:10000, Sigma-Aldrich Chemicals, St. Louis, MO, USA), cIAP2 (1:200, Biolegend, CA, USA), p-Akt1/2/3 (Ser 473) (sc-7985, 1:1000, Santa Cruz, Texas, USA), Akt1/2/3 (sc-8312, 1:1000, Santa Cruz, Texas, USA). The membranes were washed three times and incubated with HRP-conjugated secondary antibodies (1:5000, Rockland Immunochemicals, PA, USA) for 2h. Finally blots were detected by ECL chemiluminescence (Pierce, Rockford, IL, USA). Protein bands were quantified with ImageJ software (NIH) using β-actin as an internal control.

Xu J., Huang G., Zhang Z., Zhao J., Zhang M., Wang Y., Liu Z, & Lu J. (2015). Up-Regulation of Glioma-Associated Oncogene Homolog 1 Expression by Serum Starvation Promotes Cell Survival in ER-Positive Breast Cancer Cells. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 36(5).

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