Ab22665

Cell pellets were resuspended in Laemmli sample buffer (BioRad), and Western blots were performed as described previously (McClintock et al., 2006 (link)). The membranes were incubated with primary antibodies: anti-lamin A/C [kindly provided by Dr. N. Chaudhary (1/5000) (Chaudhary & Courvalin, 1993 (link)), anti-progerin antibody (clone S9, 0.1 μg mL⁻¹) (McClintock et al., 2007 (link)), anti-prelamin A antibodies (sc-6214, Santa Cruz Biotechnology, 1/1000), anti-proteasome S20 subunit C2 (ab22665, Abcam, 1/000), anti-Hsp27 (ab2790, Abcam, 1/2000), anti-ubiquitin (sc-8017, Santa Cruz Biotechnology, 1/3000), anti-LC3B (Sigma-Aldrich, 1/4000), anti-53BP1 (A300-272A, Bethyl, 1/1000)], anti-FHL-1 (sc-133580, Santa Cruz Biotechnology, 1/1000) anti-Rad51 (NBP2-32622, Novus Biological, 1/1000) anti-β-actin (Sigma-Aldrich, 1/5000) and anti-β-tubulin (Thermo Fisher, 1/2000). Then washed and incubated with a corresponding secondary antibody coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories). Proteins were visualized using a chemiluminescence detection system (ECL substrate; BioRad). Signals were analyzed with image lab software (BioRad). Protein signals were quantified by normalizing to β-actin as indicated.

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.