Extended Data Fig. 2: VPS4-mediated GSK3β sequestration during MrTAC treatment.
From: Methylarginine targeting chimeras for lysosomal degradation of intracellular proteins
a, Live-cell confocal microscopy of GSK3β-SNAP-GFP in HeLa cells incubated with Lysotracker over 60 min with 0.5 µM MrTACHaXS8. Image was captured every 10 min. Arrows note colocalization. b, Colocalization of GSK3β-SNAP-FLAG with lysosomal-associated membrane protein (LAMP-1) after MrTACHaXS8 treatment in stable HEK293 cells (0.5 µM, 3 h, mean colocalizing structures over n = 39DMSO and 54MrTAC cells; left) or transiently expressing HEK293T cells (10 µM, 3 h, mean colocalizing structures over n = 24DMSO and 45MrTAC cells; right). Arrows mark colocalization. For either experiment, graphs depict the number of colocalized GSK3β/LAMP-1 puncta within each cell. ****P < 0.0001 and ***P < 0.0010 by unpaired two-sided t-tests. c, Colocalization of HEK293T cells expressing GSK3β-SNAP-FLAG (red) with either wild-type (WT) or dominant negative (DN) VPS4-GFP after MrTACHaXS8 treatment (10 µM, 1 h). d, Colocalization of HeLa cells expressing GSK3β-SNAP-FLAG (red) with either wild-type (WT) or dominant negative (DN) VPS4-GFP after MrTACHaXS8 treatment (0.5 µM, 2 h). e, Immunoblot analysis of target protein levels of siRNA-mediated knockdown of Lamp2a, Atg7 or Vps4a (100 nM) collected on day of experiment completion (n = 3 experiments). Scale bars are 10 µm. Data are represented as means ± SEM.
