Fig. 4: Upon proteotoxic stress conditions that induce SGs, a fraction of newly synthesized SUMOylated proteins, including DRiPs, is compartmentalized at PML-NBs.
From: Loss of PML nuclear bodies in familial amyotrophic lateral sclerosis-frontotemporal dementia
A HeLa Kyoto cells were either left untreated or exposed to cycloheximide (CHX; 50 μg/ml), MG132 (20 μM), ML-792 (1 μM) alone or combined for 3 h. Then, protein extracts were subjected to SDS-PAGE followed by immunoblotting with antibodies specific for SUMO1 and SUMO2/3. TUBA4A was used as loading control. B HeLa Kyoto cells were transfected with a DNA coding for mCherry-PML. 24 h post-transfection, cells were treated with OP-puro (25 μg/ml) and MG132 (20 μM) for 3 h. Cells were fixed and immunostained with antibodies specific for SUMO1 or SUMO2/3. Nucleic acid was labeled using DAPI. Scale bar is 10 μm. C, D HeLa Kyoto cells were either left untreated (control) or exposed to OP-puro (25 μg/ml), or OP-puro (25 μg/ml) and MG132 (20 μM) for 3 h. Where indicated, cells were co-treated with CHX (50 μg/ml) to inhibit translation. Cells were fixed and subjected to click chemistry to visualize DRiPs, followed by immunostaining with antibodies specific for polyubiquitinated proteins (FK1) and either SUMO1 (C) or SUMO2/3 (D). Scale bar is 10 μm. White arrowheads show SUMO1-positive puncta; SUMO1 is used as a proxy for PML-NBs. Asterisks show nucleoli enriched for DRiPs.
