Novel MeCP2 Isoform-Specific Antibody Reveals the Endogenous MeCP2E1 Expression in Murine Brain, Primary Neurons and Astrocytes
Figure 1
Validation of the newly developed anti-MeCP2E1 antibody.
A) Schematics of MeCP2 isoforms with known functional domains. The difference in the initial amino acids of the N-terminus is highlighted. B) Schematics of the previously reported MECP2E1 (Retro-EF1α-E1) and MECP2E2 (Retro-EF1α-E2) retroviral vectors that were used for transfections (C-D) and transductions (E). C) Western blot experiments with Phoenix cell extracts from control non-transfected (NT), MECP2E1 transfected (E1-T), MECP2E2 transfected (E2-T), and MECP2E1 with peptide competition. Anti-MYC labelling was used as a positive control and ACTIN was used as a loading control. D) Western blot experiments with Phoenix cell extracts from non-transfected cells (NT), and MECP2E1 transfected cells (E1-T), probed with the anti-MeCP2E1 antibody after pre-incubation with increasing concentrations of peptide (0%, 0.1%, 1%, and 5%, of peptide as compared to the amount of antibody used). E) Immunofluorescence staining of NIH3T3 cells transduced with MECP2E1 (top row) or MECP2E2 (bottom row), with the anti-MeCP2E1 and an anti-C-MYC antibody are shown. DAPI signals are shown in blue. Note that the signals in both transduced cells are detectable with anti-C-MYC, but only transduced cells with MECP2E1 show positive signals when incubated with the anti-MeCP2E1 antibody. Scale bars represent 10 µm. MBD: methyl binding domain, ID: intervening domain, TRD: transcriptional repression domain, CTD: C-terminal domain.