Paraphenylenediamine

Cells were grown on poly-L-lysine (Wako, Osaka, Japan)-coated coverslips and fixed with 2% paraformaldehyde (Wako) in PBS for 10 min. The fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min and blocked with 5% normal goat serum (NGS; Chemicon, Temecula, CA) in PBS for 1 h. Cells were stained with primary antibodies (1% NGS in PBS) for 2 h and secondary antibodies (1% NGS in PBS) for 2 h, and then counterstained with DAPI (Roche) and mounted in PPDI [80% glycerol in PBS, 1 mg/ml paraphenylenediamine (11873580001, Roche)]. Images were recorded with a DeltaVision microscope using 60×1.40 and 100×1.35 Plan Apo objective lenses. For lamin-B staining, 5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS was used as blocking buffer, and 1% BSA in PBS was used for dilution of antibodies (Fig. S1B).

Cells were grown on coverslips coated with poly-L-lysine (Wako). After treatment with or without heat shock or MG132 as indicated in each figure caption, cells were washed with PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 1 mM KH₂PO₄), fixed in 2% formaldehyde/PBS (Polysciences) for 15 min at 37°C and then incubated with 50 mM glycine/HMK (20 mM HEPES [pH 7.5], 1 mM MgCl₂, and 100 mM KCl) for 5 min at room temperature. After permeabilization with 0.5% Triton X-100/HMK and blocking with 3% skim milk/PBS, cells were incubated with primary antibodies for 1 h at room temperature and detected with secondary antibodies conjugated with Alexa Fluor 488 or 594 (Thermo Fisher Scientific). Coverslips were mounted in PPDI (80% glycerol in PBS and 1 mg/ml paraphenylenediamine [11873580001; Roche]). DNA was counterstained with DAPI (Roche) or DRAQ5 (DR50050; Biostatus). Images were captured with an Olympus BX51 microscope (Figs 1A and B, 3A, and 5A) or FV1200 confocal microscope (Fig 1C).
The following antibodies were used as primary antibodies at the indicated dilutions: rat anti-Hsc70 (1:500; 1B5, Enzo Life Sciences) and mouse anti-Hsc70/Hsp70 (1:3,000; 1H5–1, Kose et al, 2012 (link)).