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

Figure 5.

Figure 5.

PKC mediates the phosphorylation of full-length β2PIX at Ser340 and Ser583 in cells. HEK293 cells transfected with FLAG-tagged WT β2PIX and GFP-tagged PKCγ were stimulated with 1 μm TPA in the presence or absence of 1 μm GFX, which is a pan-PKC inhibitor, or Gö6976, which is a cPKC inhibitor. FLAG-tagged β2PIX was precipitated and separated by SDS-PAGE. The total amounts of protein were determined by immunoblot analyses with an anti-FLAG antibody. GFP-tagged PKCγ or β actin was detected by anti-GFP or anti-β-actin antibodies. The phosphorylation levels of the FLAG-tagged β2PIX proteins that were determined with an anti-pT76 antibody (A; n = 2), an anti-pS340 (B; n = 4), or an anti-pS583 (C; n = 4) antibody were normalized to the phosphorylation levels of the WT. The numbers show the average relative phosphorylation levels that were normalized to prestimulation levels set as 1.00.