Liver proteins prepared in Laemmli buffer were resolved using SDS-PAGE on TGX pre-cast gels (Bio-Rad, Hercules, CA, USA) and transferred to a PVDF membrane via semi-dry transfer (Trans Blot Turbo, Bio-Rad, Hercules, CA, USA). Membranes were blocked with EveryBlot buffer (Bio-Rad Hercules, CA, USA) and incubated with the appropriate primary antibodies: ACSL1, Akt, Phospho-Akt (Ser473), GYS2, PEPCK, pFOXO1(Ser256), FOXO1, and GAPDH (Thermo Fisher Scientific, Waltham, MA, USA). After washing and incubation with an HRP-conjugated secondary antibody, protein bands were detected with an ECL chemiluminescent assay on a Bio-Rad ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA). Expression of the target protein was normalized to the GAPDH and expressed as fold changes over the control group value. Original blot pictures and their description are presented in Supplement Figures S1–S8 .
Pepck
Manufactured by Thermo Fisher Scientific
Sourced in United States
PEPCK is a core enzyme involved in gluconeogenesis, a metabolic pathway that produces glucose from non-carbohydrate precursors. It catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, a key step in the gluconeogenic process. PEPCK plays a crucial role in glucose homeostasis and is an important target for research in metabolic disorders.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Tgx precast gel, by Bio-Rad (1 mentions)
Everyblot buffer, by Bio-Rad (1 mentions)
Cells to ct kit, by Thermo Fisher Scientific (1 mentions)
Applied biosystems viia 7 instrument, by Thermo Fisher Scientific (1 mentions)
Pser727 stat3, by Cell Signaling Technology (1 mentions)
Ptyr705 stat3, by Cell Signaling Technology (1 mentions)
Socs3, by Cell Signaling Technology (1 mentions)
Sh ptp1, by Santa Cruz Biotechnology (1 mentions)
Socs3, by Thermo Fisher Scientific (1 mentions)
P jak2, by Thermo Fisher Scientific (1 mentions)
Stat3, by R&D Systems (1 mentions)
3 protocols using pepck
1
Western Blot Analysis of Liver Proteins
2
Quantifying Glucose Homeostasis Regulation
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Primary hepatocytes were prepared as described above. Cells were treated with serial diluted peptide, 10 nM insulin and 10 nM glucagon (both controls in house produced) for 4 h. After treatment, cells were lysed, and RT-PCR was performed with Cells-to-CT Kit (Ambion #4402955) according to manufacturer’s recommendation. In brief, cells were lysed with 25 µL lysis buffer with shaking at 750 rpm for 10 min before 2.5 µL stop solution was added. Reverse transcription was performed in 384 well-PCR plate. qPCR analysis was performed with Applied Biosystems ViiA7 instrument (Life technologies). G6pc and Pck1 expression in H4IIE cells were measured and normalized to Rplp0. PEPCK, G6PC and Rplp0 qPCR primers were purchased from ThermoFisher; PCK1 TaqMan Gene Expression Assays (Rn01529014 m1), G6PC TaqMan Gene Expression Assays (Rn00689876 m1), RPLP0 TaqMan Gene Expression Assays (Rn03302271 gH).
3
Immunoblotting of Hepatic Signaling
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Western blots for immunocomplexes and hepatic homogenates were performed using antibodies against phosphorylated (p) Tyr705-signal transducer and activator of transcription factor 3 (pTyr705-STAT3), pSer727-STAT3 and SOCS3 from Cell Signaling Technology (Danvers, MA), pTyr1007/1008 Janus kinase 2 (pJAK2), JAK2 and regulatory subunit of PI3K (p85) from Millipore (Temecula, CA), STAT3 from R&D Systems (Minneapolis, MN) and aquaglyceroporin-9 (AQP9), the beta chain of insulin receptor (IRb), catalytic subunit of PI3K (p110), GLUT2, long form of the leptin receptor (OBeRb), phosphoenolpyruvate carboxykinase (PEPCK) and SH-PTP1 from Santa Cruz Biotechnology (Santa Cruz, CA). The proteins were detected by chemiluminiscence using an ECL system. Quantification of the bands was carried out by densitometry using a Kodak Gel Logic 1500 Image Analysis system and Molecular Imaging software 4.0 (Rochester, NY, USA). AQP9, GLUT2, IRb, OB-Rb, p85, PEPCK, SOCS3 and SH-PTP1 were normalized with actin (Thermo Scientific, Fremont, CA), whereas pJAK2, pTyr705-STAT3 and p-Ser727-STAT3 were normalized with their respective total forms.
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