Anti cyclophilin a antibody

Whole cell lysates prepared from mouse liver, Hepa1–6 cells and mouse primary hepatocytes, and 20 µg of protein were subjected to SDS-PAGE. The separated proteins were then immunoblotted as described previously using an anti-betatrophin (Angptl8) rabbit polyclonal antibody (no. 7619, ProSci, Poway, CA), an anti-HNF-1 antibody (sc-8986 ×, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), an anti-Phospho-Akt (Ser473) (D9E) antibody (#4060, Cell Signaling Technology., Danvers, MA), an anti-GAPDH antibody (ab9485, abcam, Cambridge, UK), an anti-cyclophilin A antibody (Upstate., Lake Placid, NY) and an anti-β actin antibody (#4967, Cell Signaling Technology., Danvers, MA).

The cell extract, culture medium, and commercial human normal serum (ImmunoBioScience Corp., Mukilteo, WA) were subjected to SDS-PAGE under reducing conditions. The separated proteins were transferred to polyvinylidene fluoride transfer membranes. Membranes were incubated with an anti-annexin A2 rabbit monoclonal antibody, anti-aldolase A rabbit monoclonal antibody, or anti-cyclophilin A antibody (Cell Signaling Technology Inc., Beverly, MA, USA) at 4 °C overnight. Membranes were then washed and incubated with HRP-conjugated anti-rabbit IgG antibody (American Qualex, San Clemente, CA). After washing, blots were visualized by enhanced chemiluminescence and detected using a myECL Imager system (ThermoFisher Scientific). The same membranes were reprobed with anti-β-actin antibody (Sigma) to confirm equal loading of the proteins. All Western blot analyses were performed three times.

Samples were prepared from cell culture flasks with lysis buffer (SLB) supplemented with colorless β-mercaptoethanol (BPB), Phospho-Stop and protease inhibitor (Thermo Fisher Scientific). Cell lysates were cleared by centrifugation 20,000 rpm for 3 min. Protein concentration was measured using the Qubit Protein Assay Kit (Thermo Fisher Scientific), and 50 μg protein of each sample were loaded to a 10-well SDS, 4-12% Bis-Tris NuPage gel (Invitrogen). Proteins were then separated at 80 V for 30 min, followed by 110 V until completion. Blotting was performed with a semi-dry transfer unit on an ECL nitrocellulose membrane at 3.3 mA/1 cm²/1 h/gel. Afterwards the membrane was stained with Ponceau. The membrane was blocked in 5% milk for 1 h, then washed twice for 5 min with TBST and stained with anti-AMPK or anti-p-AMPK (Thr172) antibodies (Cell Signaling; 1:2000) in 5% BSA at 4°C and with an anti cyclophilin A antibody (Cell Signaling; 1:5000) as loading control. After three 10-min cycles of washing with TBST, the membrane was stained with the secondary antibody (anti-rabbit; DakoCytomation; 1:2000) for 1 h at room temperature, followed by another 3 wash cycles. Proteins were visualized using ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) 1:1 for 2-3 min.