Trans blot turbo nitrocellulose transfer pack

Mitochondria-enriched fraction was prepared as described previously^{10 (link)}. BMDMs were cultured in a six-well plate (1.0 × 10⁶ cells per well) and the cells were washed with PBS and re-suspended in 100 ml of mitochondrial isolation buffer (70 mM Tris, 0.25 M sucrose and 1 mM EDTA, pH 7.4), 100 ml of MES buffer (19.8 mM EDTA, 0.25 M D-mannitol and 19.8 mM MES, pH 7.4) and 4 ml of 10 mg ml⁻¹ digitonin. After 10 min of incubation, the cells were scraped and collected from three wells and centrifuged at 900g for 2 min. The supernatant was collected and centrifuged at 13,000g for 5 min to pellet the mitochondria-enriched fraction. The pellet was washed in PBS and centrifuged at 16,000g for 5 min. This fraction was then directly re-suspended in 30 ml of 1× loading buffer and boiled. The samples were then loaded into 4–20% Mini-PROTEAN TGX Precast Protein Gel (Bio-Rad), and transferred to Trans-Blot Turbo Nitrocellulose Transfer Pack (Bio-Rad). Anti-CPT2 (1:1,000 dilution; ab181114; Abcam) and anti-COX4 (1:2,000 dilution; A21348; Thermo Scientific) were used as primary antibodies and the blots were acquired and analyzed by Odyssey CLx imager (LI-COR).

Cell lysates were generated by lysis with a buffer containing 125 mM Tris (pH 6.8), 2% SDS, 20% glycerol, 100 µM PMSF, protease inhibitor mixture (Promega), and HALT™ phosphatase inhibitor cocktail (Thermo Scientific). Total protein content of the samples was assessed by BCA protein assay (Pierce). Equal amounts of protein were separated on 10% or 4%~20% Criterion gels (Bio-Rad) by SDS-PAGE. Medium and high molecular weight proteins (55-289KDa) were transferred to nitrocellulose membranes using a Trans-Blot^® Turbo™ Nitrocellulose Transfer Pack and Trans-Blot^®Turbo™ transfer system (Bio-Rad). Low molecular weight proteins (< 55kDa) were transferred to nitrocellulose membranes (Hybond; Amersham Biosciences) using a Trans-Blot SD semidry electrophoretic transfer cell (Bio-Rad). Ab-bound proteins were detected using an ECL Western blotting analysis system (Amersham Corp.), and the membranes were exposed to SRX-101A film (Konica Minolta). Densitometry analysis was performed using the UN-SCAN-IT gel (version 6.1) software. Scans of the films representing the Western blots of p-JAK1 levels, p-PI3K p55 levels, total TSC2 level, SOCS3 level and p-STAT3-Ser727 levels required use of the auto-equalize feature of CorelDraw software to optimize visualization of the bands in printed versions of the image.