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. 2019 Apr 12;14(5):882–892. doi: 10.1021/acschembio.8b01016

Figure 2.

Figure 2

Localization and function of VPS34 and SGK3 are unaffected by fusion to HaloTag7. A–C. Parental 293 (A), Halo-VPS34 (B), and SGK3-Halo (C) cells were treated for 15 min with HaloTag-TMR Ligand, and the ligand was washed out for 15 min. Cells were stained with Rab5 early endosomal marker to detect localization of HaloTag fusion proteins on endosome. Assays were performed in the presence or absence of 1 μM VPS34-IN1. D. Quantification of PtdIns3P, measured using a 2XFYVE domain probe, colocalizing to Rab5 expressing endosomes in Parental 293 and Halo-VPS34 cells. E. VPS34 was immunoprecipitated from Parental 293 and Halo-VPS34 cells, and immunoprecipitates were blotted for VPS34 complex components. F. Parental 293, SGK3-KO, and SGK3-Halo cells were treated for 1 h with either Akt inhibitor AZD5363 (3 μM), SGK3 inhibitor 14H (1 μM), or both. Cells were lysed and immunoblotted for the targets described.