Skip to main content
. 2019 Aug 21;20(10):e48014. doi: 10.15252/embr.201948014

Figure EV2. Rescue of effects of UBE2QL1 depletion on lysosomal ubiquitination (related to Fig 2).

Figure EV2

  1. Rescue of effects of UBE2QL1 depletion on lysosomal ubiquitination after damage. HeLa cells were transfected with siCtrl or siUBE2QL1 #4 for a total of 72 h. 24 h after beginning of siRNA treatments, cells were transfected with expression plasmids for UBE2QL1 wild‐type (WT) or the catalytic inactive mutant C88S (CS) tagged with HA, or GFP alone as control as indicated. Lysosomal damage was induced by 250 μM LLOMe or EtOH alone (untreated) for 3 h. Cells were fixed and processed for immunofluorescence confocal laser microscopy with antibodies specific for the HA‐tag, polyubiquitin (FK2), and LAMP1. Scale bar: 20 μm.
  2. Automated quantification of (A). FK2 and LAMP1 vesicles were identified, and the percentage of LAMP1 vesicles that were positive for ubiquitin was determined. The LLOMe‐treated siCtrl control was set to 100% for each type of overexpressed protein. The graph represents data from three independent experiments with ≥ 25 cells per condition (mean ± SD). *P < 0.05; **P < 0.01 (one‐way ANOVA with Dunnett's multiple comparison test).
  3. Western blot analysis with UBE2QL1 antibody of indicated samples related to the rescue experiment in (A). Note that the band of endogenous UBE2QL1 is only visible after long exposure. GAPDH was probed as a loading control.