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. 2017 May 31;8:15637. doi: 10.1038/ncomms15637

Figure 5. WIPI3 interacts with the TSC complex.

Figure 5

(a) Stable GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 U2OS cells were starved (3 h), and subjected to anti-GFP immunoprecipitation and anti-TSC2, anti-phospho-TSC2 (S1387), anti-TSC1 or anti-GFP immunoblotting. (b) U2OS cells were analysed by anti-TSC1 immunoprecipitation (TSC1-IP), anti-TSC1, anti-TSC2 and anti-WIPI1 (right panel), anti-WIPI2 (right panel), anti-WIPI3 (left panel) or anti-WIPI4 (right panel) immunoblotting. (c) ATG5 WT or KO MEFs expressing GFP-WIPI3 (W3) or GFP were subjected to anti-GFP immunoprecipitation and anti-TSC1, anti-TSC2 and anti-GFP immunoblotting. Conditions (3 h): fed (F) or starved (S). (d) Stable GFP-WIPI3 or GFP U2OS cells were analysed by anti-GFP immunoprecipitation and anti-TSC2 and anti-GFP immunoblotting. Conditions (15 min. to 3 h): fed (F), starved (S), starved with LY294002 (S+LY). (e) Myc-tagged TSC1 fragments (TSC1-M, TSC1-C; full length: TSC-FL) were expressed in stable GFP-WIPI3 U2OS cells and subjected to anti-GFP immunoprecipitation and anti-GFP or anti-myc immunoblotting. (f) Lysosomal fractions (no. 1–7; total protein control: TP) from stable GFP-WIPI3 U2OS cells (BafA1, 3 h) were immunoblotted using anti-GFP, anti-LAMP2, anti-TSC2, anti-WIPI2 or anti-Rab4 antibodies. (g) Stable GFP-WIPI3 U2OS cells were starved (3 h) and immunostained with anti-TSC2/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies for confocal LSM. (h) Stable GFP-WIPI3 U2OS cells were immunostained with anti-Lamp2/IgG-Alexa Fluor 633 and anti-WIPI2/IgG-Alexa Fluor 546 for confocal LSM. Conditions (3 h): fed (F), starved (S). (i) Stable GFP-WIPI3 U2OS cells with siControl or siTSC2 were fed (F) or starved (S) with or without BafA1 (3 h). Upper panel: anti-TSC2 immunoblotting, lower panel: GFP-WIPI3 puncta assessment (up to 493 cells per condition, n=4). (j) U2OS cells were subjected to immunoblotting using anti-phospho-mTOR (S2481), anti-mTOR, anti-phospho-ULK1 (S757), anti-ULK1 and anti-tubulin antibodies. Treatments: fed (F, 4 h), starved (S, 4 h), starved (3 h) and fed (1 h) (S→F). (k) Stable GFP-WIPI3 U2OS cells were immunostained with anti-mTOR/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies. Treatments: starved (S, 2 h), starved (2 h) and fed (1 h) (S→F). Co-localizations are indicated with arrows (g,h,k). Supplementary Material is available: Supplementary Fig. 5. Statistics and source data: Supplementary Data 1. Mean±s.d.; heteroscedastic t-testing; P values: *P<0.05, **P<0.01, ***P<0.001. Scale bars: 3 μm.