Figure 5. After endocytosis, SLO is ubiquitinated and sorted by the ESCRT complex into the lumen of MVBs for degradation.

A and B) Cells were incubated for 5 min at 4°C with 150 and 250 ng/mL GFP-SLO, respectively, washed and shifted to 37°C in medium with or without Ca²⁺ followed by solubilization and pulldown of ubiquitinated proteins using UBA-agarose (A) or pulldown of GFP-SLO using GFP-Trap (B). A) Ubiquitinated GFP-SLO is detected by immunoblot with anti-GFP antibodies after 1 min in the presence of Ca²⁺, and then decreases over time. Without Ca²⁺ and in control UBAmut pulldowns, only trace amounts of ubiquitinated GFP-SLO are detected. B) Ubiquitinated GFP-SLO is detected by immunoblot using anti-ubiquitin after 1 min in the presence of Ca²⁺ and then decreases over time. Without Ca²⁺, only a low amount of ubiquitinated SLO is detectable. C and D) Cells treated with control or Vps24 siRNA were exposed to GFP-SLO, followed by pulldown and immunoblot to assess GFP-SLO intracellular degradation. C) Immunoblot of GFP-Trap pulldowns shows GFP-SLO degradation between 5 and 30 min in cells treated with control, but not Vps24 siRNA. Immunoblot of cell extracts shows the depletion in Vps24 achieved with the specific siRNA, and the similar amounts of actin detected after treatment with Vps24 or control siRNA. D) Densitometric quantification of GFP-SLO at 15 and 30 min, expressed as percentage of the amount detected at t = 5 min. Results are expressed as the mean ± SEM of three independent experiments. **p < 0.01, *p < 0.05, unpaired Student’s t test. E and F) Cells treated with control or Vps24 siRNA were processed for DQ-BSA uptake as described in Figure 4C. E) Acidic vesicles containing fluorescent DQ-BSA in cells treated with control or Vps24 siRNA, exposed or not to SLO. DQ-BSA fluorescence is depicted in white and DAPI-stained nuclei in gray. Bar = 5 μm. F) Quantification of the experiment in (E) shows the enhancement in DQ-BSA fluorescence intensity induced by SLO, and the significant inhibition after treatment with Vps24 siRNA, compared to control siRNA. **p < 0.01, unpaired Student’s t-test.