Sc 6214

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.

Proteins were separated by 10% SDS-PAGE, transferred onto an Immobilon-FL membrane (Millipore) and membranes blocked with Odyssey blocking buffer (LI-COR). Membranes were incubated in Odyssey blocking buffer with antibodies against lamin A/C (1:2000, sc7292x, Santa Cruz Biotechnology; or a rabbit anti-lamin A/C antibody [44]; 1:1000), pre-lamin A (1:200, sc6214, Santa Cruz Biotechnology), lamin B1 (1:1000, sc6216, Santa Cruz Biotechnology), ZMPSTE24 (1:1000, AP2415a, Abgent) or γ-tubulin (1:10,000, T5326, Sigma-Aldrich). Proteins were detected with IRDye-800- or IRD IRDye-680-coupled secondary antibodies.

Antibodies employed for western blotting analysis or immunofluorescence labeling were as follows: anti-lamin A/C, goat polyclonal (SC-6215, Santa Cruz Biotechnology); anti-prelamin A, goat polyclonal (SC-6214 Santa Cruz Biotechnology); anti-prelamin A3, rabbit polyclonal^{26 (link)}; PPARγ rabbit polyclonal (Cell Signaling Technology); anti-UCP1 antibody (Santa Cruz); anti-TOM20 antibody (Millipore); anti-LC3 rabbit polyclonal (NB100-2220, Novus Biological); anti-P62/SQSTM1, guinea pig polyclonal (GP62-C, Progen Biotechnik); anti-p70S6 kinase and anti-phospho-p70S6 kinase (Thr389) (Cell Signaling Technology); anti-Erk 1/2 and anti-phospho Erk1/2 (Thr202/Tyr204) (Cell Signaling Technology); anti-actin, goat polyclonal (A1616, Santa Cruz Biotechnology); anti-GAPDH, mouse monoclonal (MAB374, Millipore).