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. 2020 Nov 23;17(10):2891–2904. doi: 10.1080/15548627.2020.1847798

Figure 4.

Figure 4.

GLIPR2 inhibits PtdIns3K-C1 complex lipid kinase activity. (A and B) PtdIns3P probe staining (A) and quantification (B) of PtdIns3P puncta in indicated cells. Bars represent mean ± SEM for triplicate samples (50 cells analyzed per sample). Similar results were observed in three independent experiments. n.s., not significant, ***p < 0.01; one-way ANOVA with Dunnett’s test for multiple comparisons. Scale bars, 10 µm. (C and D) Immunostaining (C) and quantification (D) of WIPI2 puncta in indicated cells. Bars represent mean ± SEM for triplicate samples (100–150 cells analyzed per sample). Similar results were observed in three independent experiments. n.s., not significant, ***p < 0.001; one-way ANOVA with Dunnett’s test for multiple comparisons. Scale bars, 10 µm. (E) In vitro pull-down assays of purified Strep-tagged PtdIns3K-C1 with wild-type GLIPR2 (WT GLIPR2) or mutant GLIPR2 (H54A E86A G102K H103A N138G) (mt GLIPR2) analyzed by SDS-PAGE and stained with Coomassie Blue. (F) In vitro pull-down assays of Strep-tagged PtdIns3K-C1 with WT GLIPR2 or mt GLIPR2, analyzed by western blotting. The left and right panels were cropped from the same gel. (G) Effect of PIK-III and CHAPS on in vitro lipid kinase activity of PtdIns3K-C1 as measured by mass spectrometry analyses. Bars represent mean ± SEM from five independent experiments. ***p < 0.001; one-way ANOVA with Dunnett’s test for multiple comparisons. (H) Effect of GLIPR2 on in vitro lipid kinase activity of PtdIns3K-C1 as measured by mass spectrometry analyses. Bars represent mean ± SEM from five independent experiments. n.s., not significant, *p < 0.05; one-way ANOVA with Dunnett’s test for multiple comparisons. (I) Effect of Tat-BECN1 on in vitro lipid kinase activity of PtdIns3K-C1 as measured by mass spectrometry analyses. Bars represent mean ± SEM from six independent experiments. n.s., not significant, ***p < 0.001; one-way ANOVA with Dunnett’s test for multiple comparisons. (J and K) Effects of Tat-BECN1 and GLIPR2 on PtdIns3K-C1 membrane association assessed using liposome flotation assays. Four fractions were collected from each sample and subjected to SDS-PAGE and immunodetection of BECN1 and PIK3C3/VPS34. Representative western blots (J) of BECN1 and PIK3C3/VPS34 in fractions with indicated treatments. Similar results were observed in four independent experiments and the relative quantification (K) of BECN1 and PIK3C3/VPS34 in liposome-containing Fraction 1. Bars represent mean ± SEM. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Dunnett’s test for multiple comparisons