Mouse anti ran

Cells were lysed in SDS-PAGE sample buffer, boiled for 5 min, and then sonicated to shear DNA. Samples were separated on 4–20% gradient gels (Mini-PROTEAN TGX; BioRad), then transferred to a nitrocellulose membrane. Membranes were blocked using 10% (vol/vol) adult bovine serum and 0.2% TritonX-100 in PBS for 20 min. The membrane was incubated with primary antibodies (anti-Sun1 (Atlas Antibodies, HPA008346, 1:1000), rabbit anti-Sun2 (Atlas Antibodies, HPA001209, 1:1000), mouse anti-Vimentin (Abcam, ab8978, 1:1000), mouse anti-Ran (BD Transduction Laboratories, 610341, 1:5000), and mouse anti-tubulin (SantaCruz Biotechnology, DM1A, 1:10,000). Primary antibodies were labeled using goat anti-rabbit HRP (ThermoFisher Scientific, G21234, 1:10,000) and goat anti-mouse HRP (Thermofisher Scientific, A24524, 1:10,000), and detected using enhanced chemiluminescence via a Bio-Rad ChemiDoc MP System (Bio-Rad, Hercules, CA, USA). Between each primary antibody, the membrane was quenched using 30% H₂0₂.

Cells were grown on glass coverslips and fixed with 3% w/v PFA in PBS for 10 min then permeabilized with 0.4% w/v Triton-X 100 in PBS for 15 min. Cells were incubated with primary antibodies for 30 min: Mouse anti-Ran (BD Transduction Laboratories, 610341, 1:100), rabbit anti-Nesprin1 (Atlas Antibodies, HPA019113, 1:100), rabbit anti-Nesprin2 (Atlas Antibodies, HPA003435, 1:50), rabbit anti-Nesprin2/4 (YenZyme; peptide antigen KKAELEWDPAGDIGGLGPLGQ, 1:25, (Neelam et al., 2015 (link))), and rabbit anti-Nesprin3 (Atlas Antibodies, HPA07740, 1:50). Primary antibodies were detected with anti-mouse AlexaFluor 488 (ThermoFisher Scientific, A11029, 1:1000), anti-Rabbit AlexaFluor 488 (ThermoFisher Scientific, A11034, 1:1000), and DNA was visualized with Hoescht 33342 (1:5000). Coverslips were mounted using 10% (w/vol) Mowiol 4–88 (Polysciences). Fluorescence images were captured using a Nikon Eclipse NiE (40×/0.75 Plan Fluor Nikon objective; 20×/0.75 Plan Apo Nikon objective) microscope at room temperature with a charge-coupled device camera (CoolSnap HQ; Photometrics, Tucson, AZ, USA) linked to a workstation running NIS-Elements software (Nikon, Melville, NY, USA). All images were processed in Adobe Photoshop CC 2017 (Adobe, San Jose, CA, USA) for cropping and brightness/contrast adjustment when applicable.

Proteins were separated by SDS-PAGE and immunoblotted with the following antibodies: rabbit anti-JUND (Santa Cruz, sc-74), mouse anti-ATF4 (Santa Cruz, sc-390063), rabbit anti-cleaved caspase-3 (Cell Signaling, 9664S), mouse anti-Tubulin (Sigma, T9026), mouse anti-Ran (B.D. 610340), goat anti-GFP (Abcam, 6673), mouse anti-RPL10A (Novus, 3G2), rabbit anti-RPL7 (Novus, NB100-2269), and rabbit anti-RPS6 (Abcam, ab40820).