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. Author manuscript; available in PMC: 2014 May 1.
Published in final edited form as: Neurobiol Dis. 2013 Jul 2;59:1–17. doi: 10.1016/j.nbd.2013.06.013

Figure 4. Characterization of phosphorylated FMRP antibody (6058).

Figure 4

A, In adult hippocampal slices, 6058 selectively labeled a band around 75kDa corresponding to the molecular weight of FMRP; intensity of this band was completely eliminated by phosphatase treatment, demonstrating that 6058 exclusively recognizes phosphoprotein. B, Adult hippocampal slices treated with DHPG (100 µM × 30 mins) were compared to untreated matched-slice controls. Intensity of 6058 immunoreactivity at 75 kDa was increased by DHPG treatment, demonstrating that 6058 exclusively recognizes relevant biologically active FMRP phosphoprotein. Quantification of DHPG-induced phosphorylation of FMRP (control 6058::total ratio: 100±39.12, n = 4, treated 6058::total ratio: 171.21±37.47, n = 4, p< 0.04, Repeated Measures ANOVA). C, 6058 immunoreactivity is absent in HEK293 cells transfected with S499A–FMRP mutant, while present when transfected with wild-type FMRP. D, FMR1 KO mice do not display immunoreactivity at 75 kDa when probed with antibody 6058 compared to controls which display robust immunoreactivity.