Anti coxii

For immunocytochemistry, fibroblasts were grown on coverslips, fixed with 100% methanol at -20°C for 10 min and were further processed for immunohistochemistry as previously described [32 ]. The following primary antibodies were used: anti-progerin S9 (1 μg/mL) [34 (link)], anti-lamin A/C (Chaudhary N, 1:500), anti-lamin B1 (sc-6217, Santa Cruz Biotechnology, 1:75), anti-γH2AX (JBW301, Millipore, 1:200), anti-53BP1 (A300-272A, Bethyl, 1:1000), anti-Rad51 (NBP2-32622, Novus Biological, 1:100), anti-Nox4 (Novus Biological, 1:1000), and anti-CoxII (Abcam, 1:1000). The secondary antibodies used were affinity-purified Alexa Fluor 488 goat or donkey IgG antibodies (Molecular Probes) and Cy3-conjugated IgG antibodies (Jackson ImmunoResearch). DAPI in Vectashield mounting medium (Vector Inc.) was used for counterstaining. The images were acquired (exposure time-matched) using an Axioplan fluorescence microscope (Carl Zeiss).

The following antibodies were used for this study: anti-Vinculin (Invitrogen, # 700,062), OxPhos Human WB Antibody Cocktail (Invitrogen, # 45-8199), OxPhos Rodent WB Antibody Cocktail (Invitrogen, #45-8099), anti-TFAM (Sigma-Aldrich, #ABE483), anti-GAPDH (Sigma-Aldrich, #G9545), anti-COXII (Abcam, #ab198286), MTCO1 Monoclonal Antibody (Invitrogen, # 459,600), goat anti-mouse IgG (H + L) HRP conjugated (Jackson ImmunoResearch), and goat anti-rabbit IgG Antibody, (H + L) HRP conjugated (Jackson ImmunoResearch).

Cell pellets were resuspended in Laemmli sample buffer (BioRad), and Western blots were performed as described previously [32 ]. The membranes were incubated with the following primary antibodies: anti-lamin A/C (kindly provided by Dr. N. Chaudhary (1:10000)) [33 (link)], anti-progerin (rabbit monoclonal, 0.1 μg/mL) [34 (link)], anti-proteasome S20 subunit C2 (ab22665, Abcam, 1/4000), anti-Ubiquitin (sc-8017, Santa Cruz Biotechnology, 1:3000), anti-LC3B (Sigma-Aldrich, 1:10000), anti-SQSTM1/p62 (ab56416, Abcam,1:2000), anti-53BP1 (A300-272A, Bethyl, 1:3000), anti-Rad51 (NBP2-32622, Novus Biological, 1:1000), anti-Nox4 (Novus Biological, 1:1000), anti-CoxII (Abcam, 1:1000), anti-Hsp27 (Abcam, 1:3000), anti-Ubiquitin (Santa Cruz Biotechnology, 1:3000), anti-pS6RP (4856, Cell Signaling, 1:1000), p4EBP1 (2855, Cell Signaling, 1:1000), anti-S6RP (Cell signaling, 1:1000), anti-4EBP1 (Cell signaling, 1:1000), and anti-β-actin (Sigma-Aldrich, 1:10000). Membranes were washed and incubated with a corresponding secondary antibody coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories). Proteins were visualized with a chemiluminescence detection system (ECL substrate; BioRad), and signals were analyzed using IMAGE LAB software (BioRad). Protein signals were quantified by normalizing to β-actin as indicated.