Chchd2 hpa027407

30 µg protein from whole-cell extracts or cellular fraction extracts was used for Western blotting analysis. Antibodies used for Western blotting were CHCHD2 (HPA027407, 1:200; Sigma-Aldrich) and GAPDH (G9545, 1:2,000; Sigma-Aldrich), SIRT1 (2493, 1:1,000; Cell Signaling Technology), SMAD4 (9515, 1:1,000; Cell Signaling Technology), p-SMAD2 (3108, 1:1,000; Cell Signaling Technology), SMAD2 (5339, 1:1,000; Cell Signaling Technology), p-SMAD3 (9520, 1:1,000; Cell Signaling Technology), SMAD3 (9523, 1:1,000; Cell Signaling Technology), histone H3 (ab1791, 1:3,000; Abcam), VDAC1 (ab15895, 1:1,000; Abcam), and β-tubulin (T4026, 1:800; Sigma-Aldrich). Quantification of Western blotting results was performed using ImageJ software.

In brief, cells were fixed in 4% PFA (Sigma-Aldrich) for 15 min, followed by a 10-min permeabilization step (0.1% Triton X-100 in PBS for internal cell markers). The blocking step was performed by incubation in 2% BSA for 30 min. Cells were incubated with primary antibodies at 4°C overnight and further incubated with secondary antibodies for 1 h. For MitoTracker dye staining, cells were cultured in the normal media with 150 nM MitoTracker red (Invitrogen) for 30 min before further fixation and immunostaining. Antibodies against the following proteins were used at the indicated dilutions: CHCHD2 (HPA027407, 1:200; Sigma-Aldrich), SOX1 (4194S, 1:200; Cell Signaling Technology), NESTIN (MAB5326, 1:200; EMD Millipore), TUJ1 (MRB-435P, 1:500; Covance), mtTFA (ab119684, 1:500; Abcam), anti–mouse IgG-FITC (1:800; Sigma-Aldrich), and anti–rabbit IgG-Cy3 (1:1,000; Jackson ImmunoResearch Laboratories, Inc.). Nuclei were labeled with DAPI (Thermo Fisher Scientific). Colocalization coefficient studies were performed using ImageJ software by calculating Manders’ colocalization coefficient, which describes the amount of colocalizing pixels of GFP using pixels generated by RFP.