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. 2014 Jul 9;34(28):9268–9280. doi: 10.1523/JNEUROSCI.4278-13.2014

Figure 3.

Figure 3.

βPIX is phosphorylated by PKCγ in vitro, in cells, and in vivo. A, In vitro phosphorylation of β2PIX. FLAG-tagged β2PIX proteins were purified and incubated with or without recombinant PKCγ in the presence of PKC activator (PS/DO/Ca2+) and [γ-32P] ATP for 20 min. The in vitro phosphorylation of β2PIX was also performed in the presence of GFX. The phosphorylated proteins were detected by autoradiography and the levels of protein expression were determined by Western blotting with an anti-FLAG antibody. The numbers show the relative phosphorylation levels that were normalized to PKCγ stimulation as 1.00 (n = 3). B, In-cell phosphorylation of β2PIX. HEK293 cells expressing FLAG-tagged β2PIX and GFP-tagged PKCγ were stimulated with 1 μm 12-O-TPA for 20 min in the presence or absence of 1 μm GFX or 1 μm Gö6976. FLAG-tagged β2PIX proteins were purified with anti-FLAG agarose resin. Phosphorylated proteins were detected by an immunoblot analysis with an anti-pSer PKC motif antibody. Protein expression was determined by Western blotting with an anti-FLAG antibody. The average relative phosphorylation levels for each experimental condition were normalized to the prestimulation signal set as 1.00 (n = 5). C, PC12 cells expressing FLAG-tagged β2PIX were incubated with 32P monosodium phosphate and stimulated with 1 μm 12-O-TPA in the absence or presence of 1 μm GFX. FLAG-tagged β2PIX proteins were purified with anti-FLAG agarose resin. Phosphorylated proteins were detected by autoradiography, and protein expression was determined by immunoblots with an anti-FLAG antibody. D, The same amount of samples of total fraction and the P2 synaptosomal fraction were immunoblotted by anti-PSD95 antibody as a postsynaptic marker, anti-VMAT2 antibody as a presynaptic marker, and anti-PKCγ antibody. E, the P2 synaptosomal fraction was stimulated with 2 μm 12-O-TPA in the absence or presence of 2 μm GFX. βPIX proteins were purified with anti-βPIX antibody. Phosphorylated proteins were detected by phospho-Abs. The numbers show the relative phosphorylation levels that were normalized to pretreatment as 1.00 (n = 3).