Fig 1: Biochemical characterisation of VPS34 complexes reconstituted with VPS15 phosphomutants VPS15 KO cells were transfected with VPS34, Beclin1, ATG14 and with either wild type (WT), 6SA or 6SE VPS15 and starved for 60 min as indicated before lysis and VPS34 complex coimmunoprecipitation via VPS15. Compared to WT, the in vitro lipid kinase activity of VPS34 complex I incorporating 6SA or 6SE VPS15 was reduced on ~800 nm large unilamellar vesicles (LUVs). PI3P was detected via a GST overlay assay using the GST-PX probe. Reaction mixtures were analysed by Western blot (right-hand side) to show VPS34 complex amounts used, a representative experiment of three independent repeats is shown. Dashed lines indicated where blots were cropped for presentation.HEK293A were cotransfected with VPS34, Beclin1, ATG14, UVRAG and with either empty vector (EV) or wild type (WT), 6SA or 6SE VPS15. After starvation for 30 min, cells were lysed and relative VPS34 complex component immunopurification via VPS15 was assessed via immunoblotting. Quantifications represent mean ± SEM, n = 3.No difference in in vitro lipid kinase activity on 100 nm LUVs was detected with VPS34 complex I incorporating either VPS15 WT, 6SA or 6SE. ADP-Glo kinase assay (Promega V6930) was used to assess lipid kinase activity and mean ± SD from 4 technical repeats are shown. Data reflect representative data from 1 of 3 independent repeats.The membrane binding affinity of VPS34 CI incorporating either WT, 6SA or 6SA VPS15 on 100 nm LUVs (large unilamellar vesicles) was assessed via liposome floatation assay. Complexes segregating to the top (liposome-bound) and bottom (liposome-unbound) of a sucrose gradient were separated by SDS–PAGE and Coomassie-stained gels are shown. When the percentage of VPS15 and VPS34 in the top fraction was quantified, none of the differences between WT, 6SA or 6SE were significant. In = input; mean ± SD, n = 3. Data information: (B) *P < 0.05, n.s. not significant, [one-tailed ANOVA]. (C, D) n.s. not significant, [Student’s t-test].