Fig. 4

STYK1 promotes cell membrane-associated GSK3β sequestration in an ESCRT-dependent manner. a Confocal microscopy images of mCherry-tagged STYK1 and EGFP-tagged Rab5 and its Q79L mutant in HeLa cells. Scale bar: 10 μm. b, c Representative immunofluorescence images of RFP-tagged GSK3β and EGFP-tagged Rab5 Q79L mutant, and the quantification of the amount of GSK3β within Rab5⁺ endosomes in response to Wnt3a treatment after STYK1 depletion in PANC-1 cells (n = 10). Scale bar: 10 μm. d, e Representative immunofluorescence images of RFP-tagged GSK3β and EGFP-tagged Rab5 Q79L mutant, and the quantification of the amount of GSK3β within Rab5⁺ endosomes with or without Wnt3a treatment after STYK1 overexpression and β-catenin knockdown in human bone osteosarcoma epithelial (U2OS) cells (n = 10). Scale bar: 10 μm. f Representative immunofluorescence images and the quantification of the co-localization between Rab7 and RFP-tagged GSK3β upon Wnt3a treatment (n = 3). Scale bar: 10 μm. g Western blot analysis of p-LRP6 S1490, GSK3β, and the active form p-GSK3β Y216 levels after STYK1 depletion using plasma membrane separation assays in PANC-1 cells. h The level of p-GSK3β Y216 in the LRP6 immunoprecipitate by western blot. i, j GSK3β puncta formation in response to Wnt3a treatment was assessed by immunofluorescence imaging and quantified in PANC-1 cells with HRS/VPS24 depletion (n = 10). Scale bar: 10 μm. k The level of β-catenin after STYK1 overexpression in the PANC-1 cell lysates with HRS or VPS24 knockdown by western blot assays