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. 2014 Dec;34(24):4379–4388. doi: 10.1128/MCB.00740-14

FIG 4.

FIG 4

Hypoxia-induced phosphorylation of Atg9A at S761 mediates 14-3-3ζ binding. (A) HA-Atg9A was overexpressed in HEK-293 cells, and cells were treated with normoxia or hypoxia for 12 h. HA-Atg9A was immunoprecipitated and analyzed by LC-MS/MS. The signal intensities of the S761 phosphopeptide were measured by using software described in Materials and Methods. (B) HA-tagged WT Atg9A, an S761A mutant, and an S656A/S761A double mutant (DM) were overexpressed in HEK-293 cells, and cells were treated with normoxia or hypoxia for 12 h. Proteins were subjected to co-IP and immunoblotting for endogenous 14-3-3ζ. (C) HA-tagged WT Atg9A and the S761A mutant were treated as described above for panel B and then immunoblotted with an anti-14-3-3ζ phosphomotif antibody. (D) HA-tagged WT Atg9A and the Atg9A S761A mutant were overexpressed in U2OS cells, and cells were then treated and analyzed as described above for panel C. (E) HEK-293 cells treated with or without hypoxia for 12 h were lysed and immunoblotted with the anti-14-3-3ζ phosphomotif antibody to detect phosphorylation of endogenous Atg9A. (F) HA-tagged WT Atg9A and the S761A mutant were overexpressed in HEK-293 cells and subjected to the indicated treatments for 12 h. Immunoprecipitated proteins were immunoblotted with the anti-14-3-3ζ phosphomotif antibody.