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. 2018 Jan 10;38(2):278–290. doi: 10.1523/JNEUROSCI.1649-17.2017

Figure 5.

Figure 5.

Phosphorylation of CSPα at Ser10 and Ser34 increases the ubiquitination/degradation of SNAP25 in PC12 cells. A, Isolated FLAG-tagged SNAP25 by anti-FLAG Ab from PC12 cells, transfected with GFP-tagged CSPα (WT and Ser/Ala mutants) and FLAG-tagged SNAP25 and HA-tagged ubiquitin, was immunoblotted by anti-ubiquitin Ab. The ubiquitin levels of FLAG-tagged SNAP25 from PC12 cells transfected with GFP-tagged CSPα (WT and Ser/Ala mutants) were normalized with respect to WT, as shown in the bar graph. The results are expressed as mean ± SEM (n = 5, *p < 0.05 vs WT, 1-way ANOVA with post hoc Dunnett's test). The star indicates the ubiquitinated FLAG-tagged SNAP25. B, Endogenous SNAP25 protein levels were measured in PC12 cells transfected with GFP-tagged CSPα (WT, Ser/Ala mutants, and Ser/Asp mutant) by using immunoblot for anti-SNAP25 Ab. SNAP25 protein levels were normalized with respect to WT, as shown in the bar graph. The results are expressed as mean ± SEM (Control, n = 12; WT, n = 13; S10A, n = 7; S34A, n = 7; S10A/S34A, n = 13; S10D/S34D, n = 8; *p < 0.05 vs WT, 1-way ANOVA with post hoc Games–Howell's test).