Glyceraldehyde 3 phosphate dehydrogenase gapdh

Rabbit monoclonal antibodies against ITGB3 (#4702) was purchased from Cell Signaling Technology. Mouse monoclonal antibodies against β-actin (#AC010), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#AC002) and horseradish peroxidase-conjugated secondary antibodies were purchased from ABclonal Technology, China.

Fetal bovine serum (FBS), Dulbecco's Modified Eagle's Medium (DMEM) and penicillin-streptomycin solution were purchased from Gibco Laboratory (Carlsbad, CA, USA). Trizol reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratorise (Kumamoto, Japan). catechin (> 97%), 3-isobutyl-1-methylxanthine (IBMX), insulin, dexamethasone (DEX), TNF-α and Oil Red O were ordered from Sigma-Aldrich Company (St. Louis, MO, USA). HyPure^TM Molecular Biology Grade Water, phosphate buffer solution (PBS) were purchased from Hyclone (Waltham, MA, USA). The reagents for electrophoretic mobility shift assays were from SunShine Biotechnology (Hangzhou, China). RevertAid First Strand cDNA Synthesis Kit was from Thermo Scientific (Waltham, MA, USA). FastStart Universal SYBR Green Master (Rox) was from Roche (Basel, Switzerland). Antibodies for NF-κB-p65, p-NF-κB-p65 (Ser276) were from Abcam Inc. (Cambridge, MA); SIRT1, p-SIRT1 (Ser27), FOXO3a, p-FOXO3a (Ser253) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from ABclonal Biotechnology (Woburn, MA, USA); AMPK and p-AMPK(Thr172) were from Cell Signaling Technology (Danvers, MA, USA). Cell culture dishes and multi-well plates were supplied by Nunc (Denmark).

First, cells were lyzed on ice in a radioimmunoprecipitation (RIPA) buffer with a protease inhibitor then quantified by quinolinic acid (BCA) assay. Second, SDS-PAGE isolated a total of 50 μg of protein lysate, which was then transferred to a PVDF membrane (Millipore Corp, Billerica, MA, USA). Third, the membrane was blocked with 5% fat-free milk, then incubated with PGK1 (ABclonal Biotech Co., Woburn, MA, USA, 1:100), MYC (ABclonal Biotech Co., Woburn, MA, USA, 1:100), LDHA (ABclonal Biotech Co., Woburn, MA, USA, 1:100), HK2 (ABclonal Biotech Co., Woburn, MA, USA, 1:100), GLUT4 (ABclonal Biotech Co., Woburn, MA, USA, 1:100), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ABclonal Biotech Co., Woburn, MA, USA, 1:1000) at 4 °C overnight. After that, the membranes were incubated with secondary antibodies. Then, ECL test reagent (Fudebio, China) was used to show the bands.