Fig. 6

AP2-mediated STYK1 internalization enhances GSK3β sequestration through autophagy activation. a Comparative analysis of mammalian STYK1 N-terminal sequences reveals high conservation of two putative AP sorting motifs (highlighted). b, c The interaction between Flag-tagged AP1/2 μ subunits and HA-tagged STYK1 was analyzed by co-IP. d, e The interaction between Flag-tagged AP1/2 μ subunits and HA-tagged wild-type STYK1 and its SS1, SS2, and SS3 mutants were analyzed by co-IP using indicated antibodies. f Co-localization of mCherry-tagged wild-type STYK1 and its SS1, Y191F mutants with EGFR. Scale bar: 10 μm. g, h Representative immunofluorescence images of RFP-tagged GSK3β and EGFP-tagged Rab5 Q79L mutant, and the quantification of the amount of GSK3β within Rab5⁺ endosomes upon wild-type STYK1 and its SS1, SS2, SS3, Y191F and Y191D mutants transfection in U2OS cells. Scale bar: 10 μm. i Protein levels of autophagy-related genes LC3 and p62, and Wnt target genes(CyclinD1/C-myc/Axin2) in PANC-1 cells with or without Dynasore treatment. j The green/red fluorescence intensity ratio of 7TGC signals was quantified in PANC-1 cells transfected with wild-type STYK1 and its mutants (SS1, SS2, SS3, Y191F, and Y191D, n = 5). Scale bar: 100 μm. k The levels of C-myc, Axin2, and LC3 were analyzed with or without EGF (100 ng/mL), or CQ (100 μM) treatment. l The levels of C-yclinD1, Axin2, and LC3 in wild-type STYK1 and its SS1, SS2, SS3, Y191F, and Y191D mutants transfected PANC-1 cell lysates were analyzed with or without CQ (100 μM) treatment. Data were represented as mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001