JCS -- Hausser et al. 119 (17): 3613 Figure IG1

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Fig. 1. PKD-mediated phosphorylation of PI4KIIIß at Ser294 promotes binding of 14-3-3 proteins. (A) Sequence alignment of the PKD consensus motif in PI4KIIIß from various species demonstrates conservation of a putative 14-3-3 binding motif. Phosphorylation of the serine is predicted to be required for recognition. (B) A Flag-tagged human wildtype PI4KIIIß expression vector was transiently transfected into HEK293 cells. Lysates were incubated with GST-14-3-3ß, ${epsilon}$ , ${gamma}$ , ${eta}$ , ${sigma}$ , ${tau}$ , ${zeta}$ or GST coupled to Glutathione sepharose beads and bound proteins were separated by SDS-PAGE. Western blotting using a Flag-specific antibody detected the PI4KIIIß. PI4KIIIß expression was verified by immunoblotting of total cell lysates (TCL) with Flag-specific antibody. (C) Left panel: HEK293 cells transiently transfected with Flag-PI4KIIIß were treated for 1 and 2 hours with 100 nM OA solubilized in dimethyl sulfoxide. Total cell lysates (TCL) were immunoblotted and probed for phosphorylation of Ser294 with the PKD pMOTIF antibody, expression of Flag-PI4KIIIß was controlled with anti-Flag antibodies. The solvent dimethyl sulfoxide alone had no effect on the Ser294 phosphorylation. Right panel: Lysates of HEK293 cells transiently transfected with Flag-PI4KIIIß wildtype or the S294A mutant were subjected to Western blot and probed with the anti-PKD pMOTIF antibody. Expression of Flag-PI4KIIIß was controlled with anti-Flag antibodies. (D) Flag-tagged human wildtype and mutated PI4KIIIß expression vectors were transiently transfected into HEK293 cells. Cells were treated with 100 nM OA for 2 hours. Lysates were incubated with Glutathione beads coupled to GST-14-3-3 ${tau}$ and bound proteins were separated by SDS-PAGE. Western blotting using a Flag-specific antibody detected PI4KIIIß. The expression level of PI4KIIIß was verified by immunoblotting of TCL. (E) Left panel: Flag-tagged wildtype and S294A PI4KIIIß expression vectors were transiently transfected into HEK293 cells along with HA-tagged 14-3-3 ${tau}$ , Glu-Glu-tagged 14-3-3 ${gamma}$ and 14-3-3 ${zeta}$ , respectively. PI4KIIIß was precipitated using Flag-specific antibodies and protein complexes immunoblotted with Flag- and 14-3-3-specific antibodies. Total cell lysates were analysed in parallel to estimate expression levels. Right panel: Flag-tagged wildtype and S294A PI4KIIIß expression vectors were transiently transfected into HEK293 cells. 14-3-3 proteins were precipitated using 14-3-3-specific antibodies and protein complexes immunoblotted with Flag- and 14-3-3-specific antibodies. Total cell lysates were analysed in parallel to estimate expression levels. PD, pulldown.