Ab171978

Fibroblasts were lysed in 50 mM Tris–HCl pH 7.5, 250 mM NaCl, 0.1% Triton-X-100, 1 mM EDTA, 50 mM NaF, 0.1 mM Na₃VO₄, 1× Protease inhibitor/No EDTA and LCLs in 50 mM Tris–HCl pH 7.5, 50 mM KCl, 0.1% NP40, 5 mM EDTA, 50 mM NaF, 0.1 mM Na₃VO₄, 1× Protease inhibitor/No EDTA^{43 (link)}, sonicated with microtip (10 s 25% amplitude; Sonics Vibra-Cell VCX 130), cell debris removed by centrifugation at 15,000 × g for 20 min at 4 °C and proteins assayed using BCA assay kit (ThermoFisher). 8 µg protein of each sample was resolved on 4–12% Bis–Tris protein gels (ThermoFisher), transferred to nitrocellulose membranes and western blotted with rabbit monoclonal anti-TIMMDC1 antibody (1:2000; ab171978; Abcam) or anti-β-Tubulin (1:5000; Ab6046; Abcam) and polyclonal goat anti-rabbit IgG/HRP (1:1000; P0448; Dako) as secondary antibody. Signal was detected by Clarity Western ECL Substrate (Bio-Rad) and captured using Gel Documentation System (Bio-Rad). All blots and gels shown are derived from the same experiments and were processed in parallel.

Total fibroblast cell lysates were subjected to whole protein quantification, separated on 4–12% precast gels (Lonza) by SDS–polyacrylamide gel electrophoresis (PAGE) electrophoresis and semi-dry transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences). The membranes were blocked in 5% non-fat milk (Bio Rad) in TBS-T (150 mM NaCl, 30 mM Tris base, pH 7.4, 0.1% Tween 20) for 1 h and immunoblotted using primary antibodies (1:1,000 dilution) against CLPP (Abcam, ab56455), MCOLN1 (Abcam, ab28508), MT-ND5 (Abcam, ab92624), NDUFA13 (Abcam, ab110240), NDUFB3 (Abcam, ab55526), NDUFB8 (Abcam, ab110242), TIMMDC1 (Abcam, ab171978) and UQCRC2 (Abcam, ab14745) for 1 h at RT or ON at 4 °C. Signals were detected by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (Jackson Immuno Research Laboratories, Code: 111-036-045 and Code: 115-036-062, respectively, 1:5,000 dilution) for 1 h and visualized using ECL (GE Healthcare Life Sciences).