Amersham ecl reagent

Proteins in SDS buffer were denatured at 95 °C for 5 min, separated by SDS-PAGE, transferred onto PDVF membranes, blocked with 2% milk in TBST for 30 min, and probed with anti-COL6A1 (GeneTex, Irvine, CA, USA, cat. GTX109963, 1:1000) or anti-Vinculin (Sigma, St. Louis, MO, USA, cat, V9131, 1:1000). After washing four times with TBST, membranes were incubated with 1:2000 diluted HRP-conjugated secondary antibodies (Cytiva, Emeryville, CA, USA) for 30 min at RT, washed four times again with TBST, and visualized with Amersham ECL reagents (Cytiva).

Protein samples were isolated from tissue and cells using the Minute total protein extraction kit (Invent, SD001). Separated proteins were transferred to polyvinylidene difluoride membranes (Millipore Sigma Co., Ltd., Burlington, MA, United States). The membranes were probed with antibodies against SIRT3 (10099-1-AP, 1:500), LCAD (17526-1-AP, 1:300), Complex I (12444-1-AP, 1:1,000), Complex III (14865-1-AP, 1:1,000), Complex IV (18443-1-AP, 1:2,000), FAT/CD36 (18836-1-AP, 1:500), CPT-1β (22170-1-AP, 1:500), and GADPH (60004-1-Ig, 1:5,000) purchased from Proteintech as well as with an antibody against MTTP from Absin (abs136111a, 1:1,000) and antibodies against VDR (sc-1008, 1:1,000) and GLUT4 (sc-53566, 1:500) purchased from Santa Cruz. Following overnight primary antibody incubation, membranes were incubated with a secondary antibody (ZSGB-Bio, Beijing, China) for 1 h. The protein bands of interest were detected using Amersham ECL reagents (Cytiva, Little Chalfont, United Kingdom).

Tissue lysates were prepared by homogenizing intestines in ice cold protein lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100, 10% glycerol) using a Tissue Lyser II (QIAGEN; Hilden, Germany) for 12 cycles of 30 s at 30 Hz. Protein levels of tissue lysates were normalized by a BCA kit (Thermo Fisher Scientific) (Weigmann et al., 2007 (link)). After lysed in SDS-PAGE loading buffer and boiled at 95°C for 5 min, proteins were separated by SDS polyacrylamide electrophoresis using 10% gel at 120 V for 90 min, and separated proteins were transferred in to nitrocellulose membrane by doing wet transfer at 100 V for 30 min. Membranes were later blocked with 5% milk and incubated with primary antibody at 4°C for overnight with shaking. Then, membranes were incubated with secondary antibody at room temperature for 1 h with shaking and proteins were detected by Amersham ECL reagent (Cytiva).