Fig. 3: Elevated OTUD1 protein forms aggresome and re-localize ASK1 in the cytoplasm.

a IB analysis of input and IP products from 293T cells transfected with Flag-OTUD1 (Flag-WT) or Flag-C320A, ubiquitin-myc (Ubi-myc) or Ubi-K48O-myc, and HA-ASK1. The cell lysates were treated with protease inhibitor MG132 prior to Co-IP experiment. WT, OTUD1-WT; C320A, OTUD1-C320A. b Two dox-inducible EGFP-OTUD1-WT and EGFP-OTUD1-C320A expression system were established to assess the effect of OTUD1 on ASK1 protein levels. Dox, doxorubicin. c Representative immunofluorescence images of 293T cells transiently transfected with EGFP and EGFP-OTUD1 WT or EGFP-C320A plasmids. Scale bar, 25 μm. d Representative images of fluorescence recovery at prebleach (0 s) and postbleach (30 s, and 90 s). Scale bar, 10 μm. e Representative immunofluorescence images of indicated 293T cells treated with NOCO. Scale bar, 25 μm. NOCO, nocodazole. f Immunofluorescence was performed to observe the co-localization of EGFP-OTUD1 and HDAC6 (aggresome biomarker). Scale bar, 25 μm. g Representative immunofluorescence images of 293T cells co-transfected with mcherry-ASK1 and EGFP-OTUD1 or EGFP-C320A plasmid. Scale bar, 25 μm. h A dox-inducible 293T EGFP-OTUD1 expression system was generated. The resultant cells were transfected with mcherry-ASK1 and treated with doxorubicin (DOX) (1 μM) for 24 h before the media was changed. Subsequently, the aggregation of OTUD1 and ASK1 was observed by Laser Scanning Confocal Microscope at 0, 12 h, 24 h, and 48 h after DOX (1 μM) was removed. Scale bar, 5 μm. Dox doxorubicin. Representative of n = 3 independent experiments (a–h). Source data are provided as a Source Data file.