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. 2021 Mar 15;118(12):e2016730118. doi: 10.1073/pnas.2016730118

Fig. 5.

Fig. 5.

Phosphorylation of Ykt6 at the evolutionary conserved S174 plays an important role in the secretory pathway. (A) FACS analysis depicting the percent of FKBP-GFP–positive cells over time upon overexpression of mCherry, N-terminal mCherry-tagged of WT Ykt6, phosphoablative mutant (S174A), and phosphomimetic mutant (S174D) of YKT6 post addition of solubilizer. (B) Representative IF images of PC4 cells expressing FKBP-GFP reporter, transiently transfected with HA-tagged WT Ykt6 or indicated phospho-mutants of Ykt6 and immunostained with TGN-46 for trans-Golgi or cotransfected with mCherry-Sec61 for the ER (ER-RFP). (C) Colocalization analysis based on Mander’s correlation coefficient for FKBP-GFP reporter retained in trans-Golgi post 120 min of solubilizer in cells transfected with HA-tagged WT Ykt6 or indicated phospho-mutants of Ykt6. (D) Colocalization analysis based on Mander’s correlation coefficient for FKBP-GFP reporter retained in ER post 120 min of solubilizer in cells transfected with HA-tagged WT Ykt6 or indicated phospho-mutants of Ykt6. N = ∼900 cells, ****P < 0.0001. One-way ANOVA, Tukey’s test. (E) Representative Western blot for LC3-II from HEK293T transiently overexpressing GFP and N-terminal GFP-tagged fusion of WT Ykt6 or phospho-mutants. Cells were treated for 2 h with 200 nM Bafilomycin A1 (Baf-A1) in growing conditions (fresh 10% fetal bovine serum and 4.5% glucose growth medium) or in starvation conditions (1× Hank’s Balanced Salt Solution supplemented with 250 nM Torin-1 and 10 mM Hepes). Actin serves as a loading control. (F) Autophagic flux (defined as [LC3-II Baf-A1] – [LC3-II]) from Western blot in A of starvation conditions. n = 6; *P < 0.05, **P < 0.01, ****P < 0.0001. Two-way ANOVA, uncorrected Fisher’s LSD test. (G) Autophagic flux based on fluorescence intensity of GFP-LC3-II foci of overexpressed GFP-LC3 from HeLa cells expressing WT human Ykt6 and phospho-mutants, under starvation conditions with Baf-A1. (H) Representative IF images of overexpressed GFP-LC3 and endogenous Lamp-1 colocalization from HeLa cells expressing WT human Ykt6 and phospho-mutants, under starvation conditions with Baf-A1.