Cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific), and the lysate was clarified by centrifugation at 15,000 rpm for 20 min at 4 °C. The protein concentration of the lysate was measured using a bicinchoninic acid protein assay (Thermo fisher scientific#23225). A sample buffer containing 2-mercaptoethanol was added to the total protein and heated at 95 °C for 5 min. The whole-cell lysate protein was resolved via (4–10%) SDS-PAGE unit and blotted onto polyvinylidene difluoride membranes (PVDF# BIO-RAD). Membranes were blocked in 5% bovine serum albumin or skimmed milk. Specific proteins were detected by incubation with appropriate primary and secondary (BIO-RAD) (horseradish peroxidase-conjugated) antibodies in TBST containing 5% bovine serum albumin. pCREB/CREB, pMAPK/MAPK, and β-actin were purchased from Cell Signaling Technology and TXNIP from Novus Biologicals. Antibodies used are provided in Table S3 .
Pcreb creb
Manufactured by Cell Signaling Technology
PCREB/CREB is a lab equipment product from Cell Signaling Technology. It is used to detect and measure the levels of CREB (cAMP Response Element Binding protein) and its phosphorylated form, PCREB, in cellular samples.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Txnip, by Novus Biologicals (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Nampt, by Abcam (1 mentions)
2 protocols using pcreb creb
1
Western Blot Analysis of Protein Expression
2
Quantifying Protein Signaling in Brain Regions
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The PFC and HIP regions of the brain were immediately placed on ice after dissection. After sonication, total protein was extracted and quantified with a kit. Next, 60 μg of total protein was used for electrophoretic analysis for 2 h and transferred to a PVDF membrane at 100 V for 2 h under cooling conditions. pCREB/CREB (1:5000, Cell Signaling Technology), NAMPT (1:500, Abcam), and the membranes were first treated with GAPDH (1:10,000) overnight, followed by incubation with the secondary antibody conjugated to HRP (KangChen Bio-tech, Shanghai, China). Finally, the signal intensity was evaluated with Image J.
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