Hpa017340

Cells were grown on glass coverslips and fixed in 3% (wt/vol) PFA/PBS for 10 min and permeabilized by 0.4% (wt/vol) Triton X-100/PBS for 15 min. For immunofluorescence of HSP90, cells were fixed in 3% PFA for 10 min, then in −20°C methanol for 10 min, and permeabilized by 0.4% Triton X-100/PBS for 15 min. Cells were labeled for 1 h in primary antibodies: rabbit anti–BAF (1:100; ab129184; Abcam), rabbit anti-pBAF (specific for phosphorylated BAF; 1:200; generous gift from Robert Craigie, National Institutes of Health, Bethesda, MD), rabbit anti–LEMD2 (1:100; HPA017340; Atlas Antibodies), and mouse anti–HSP90 α/β (1:100; sc-13119; Santa Cruz Biotechnology). Primary antibodies were detected using Alexa Flour 488–conjugated goat anti–rabbit (1:1000; A21235; Thermo Fisher Scientific), Alexa Fluor 568–conjugated goat anti–mouse (1:1000; A11036; Thermo Fisher Scientific) and Hoescht dye 33342 to detect DNA. Coverslips were mounted using 10% (wt/vol) Mowiol 4-88 (Polysciences). Epifluorescent images were captured using a Nikon Eclipse NiE (40×/0.75 Plan Fluor Nikon objective; 20x/0.75 Plan Apo Nikon objective) microscope at room temperature with a charge-coupled device camera (CoolSnap HQ; Photometrics) linked to a workstation running NIS-Elements software (Nikon). All images were processed in Adobe Photoshop CS6 for cropping and brightness/contrast adjustment when applicable.

Protein expression was analyzed from total cell lysate. 1.2 × 10⁶ cells were lysed in SDS/PAGE sample buffer (200 µl 2× sample buffer, 245 µl H₂O, 50 µl SDS [20% wt/vol], and 5 µl DTT), boiled for 5 min, and sonicated to shear DNA. Proteins were separated on 4–20% gradient gels (Mini-PROTEAN TGX; Bio-Rad) and transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked with 10% (vol/vol) adult bovine serum and 0.2% Triton X-100 in PBS for 30 min, and then incubated with appropriate primary antibodies. All primary antibodies were used at 1:1,000 dilution unless otherwise noted. Rabbit anti-BAF (ab129184; Abcam), rabbit anti–lamin A/C (2032S; Cell Signaling Technology), rabbit anti–LEMD2 (HPA017340; Atlas Antibodies), rabbit anti-Emerin (2659S; Cell Signaling Technology), rabbit anti-Ankle2 (ab225905; Cell Signaling Technology), and rabbit anti-Chmp7 (16424-1-AP; Proteintech) were used to verify siRNA knockdown efficiency. Mouse monoclonal anti-tubulin was used as a loading control (1:2,000; sc-32293; Santa Cruz Biotechnology). Primary antibodies were detected using HRP-conjugated anti-rabbit (1:5,000; G21234; Thermo Fisher Scientific) or anti-mouse (1:5,000; F21453; Thermo Fisher Scientific) antibodies. The signals from antibodies were detected using enhanced chemiluminescence via the Bio-Rad ChemiDoc MP Imaging System.