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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: J Neurochem. 2014 Feb 12;129(4):743–752. doi: 10.1111/jnc.12657

Figure 3. A phospho-specific antibody identifies Sp4 S770 as a site of phosphorylation regulated by membrane depolarization.

Figure 3

A. 293T cells were transfected with FLAG-Sp4 or an empty vector and lysates were treated with or without lambda phosphatase (PPase) before being separated by 6–12% PAGE and immunoblotted for pSp4 and Sp4. B. Cerebellar lysates from wild-type and Sp4 hypomorph mice were separated by 6% PAGE and immunoblotted with antisera specific for phospho-Sp4 S770 (pSp4), Sp4, and RNA polymerase II as a loading control. C. Cerebellar sections from adult rat brains were subjected to immunohistochemical staining using antibodies to Sp4, pSp4, and pSp4 co-incubated with peptide antigen as indicated. ML – molecular layer; PCL – Purkinje cell layer; GCL – granule cell layer. Scale bar is 50μm. D. CG neurons were exposed to depolarizing or resting conditions for 1 hour and lysates were subjected to phosphatase treatment, separated by 10% PAGE, and analyzed by Western blot using pSp4 and total Sp4 antibodies. Top - representative immunoblot. Bottom - quantification of the ratio of phosphorylated Sp4 relative to total Sp4 and normalized to the control condition. N=3. **p<0.01, *p<0.05, ANOVA (F=47.54; p<0.0001).