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2 protocols using phospho akt2 ser474

1

Comprehensive Western Blot Antibody Panel

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The following primary antibodies were used for western blotting at 1:1000 dilution: Ago2 (MBL CM004-3), Ago1 (MBL RN028PW), Glucokinase (Abcam ab88056), β-Actin (Sigma A1978), GAPDH (Abcam ab8245), α-Tubulin (Sigma, T6557), Glucose-6-phosphatase (Abcam ab83690), Glycogen synthase (Cell Signaling, #3893), Insulin receptor (Cell Signaling, #3025), Akt2 (Cell Signaling, #2964), phospho-Akt2 (Ser474) (Cell Signaling, #8599), FoxO1 (Cell Signaling, #2880), phospho-FoxO1 (Cell Signaling, #9464), Acetyl-CoA Carboxylase (Cell Signaling, #3676), phospho-Acetyl-CoA Carboxylase (Cell Signaling, #11818), Fatty Acid Synthase (Cell Signaling, #3180), AMPKα (Cell Signaling, #2532), and phospho-AMPKα (Cell Signaling, #2535). Image densitometry of 16-bit TIF images for all western blots was performed using ImageJ.
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2

Western Blot Analysis of Cell Signaling

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Immunoblots were done as previously described (Jornayvaz et al. 2012) . Membranes were incubated overnight with primary antibodies for phospho-Akt2 (Ser 474 ) (Cell Signaling Technology, Danvers, MA, USA), phosphoenolpyruvate carboxykinase (PEPCK; Abcam, Cambride, MA, USA), pyruvate carboxylase (PC; Abcam), uncoupling protein 1 (UCP1; Santa Cruz Biotechnology), C/EBP homologous protein (CHOP; Cell Signaling Technology), IgH chain binding protein (BIP; Cell Signaling Technology), phospho-eIF2a (Cell Signaling Technology), or phospho-JNK (Cell Signaling Technology). After further washings, membranes were incubated with HRP-conjugated secondary antibody (Bio-Rad) and visualized by ECL substrate (Pierce, Rockford, IL, USA). Membranes were stripped and reblotted with anti-total Akt antibody (Cell Signaling Technology), total eIF2a (Cell Signaling Technology), total JNK (Cell Signaling Technology), or glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology). Bands were then quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).
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