Expression of Braf35 inhibits neuronal differentiation. (A) P19 cells were transfected with a GFP expression vector and with expression vectors encoding the indicated proteins or empty vector. Three days after transfection, cells were analyzed by TuJ1 immunostaining (A1–A6) and GFP expression (A7–A12). A13–A18 are overlay images. (Scale bars, 10 μm.) (B) TuJ1-positive cells were scored as a percentage of GFP-positive transfected cells. (C) Diagram showing regions occupied by proliferating progenitors (ventricular zone; VZ) and postmitotic neurons (mantle zone; MZ) in HH21 chicken embryo spinal cord. (D) The neural tube of chicken embryos was electroporated with constructs expressing the proteins indicated at the top of each panel or empty vector. GFP was used to monitor electroporation. Thirty hours postelectroporation, embryos were immunostained for TuJ1 and DAPI. (Scale bars, 50 μm.) (E) Quantification of data presented in D as a percentage of TuJ1-positive cells per total number of GFP-positive cells. (B and E) Data are the average of three independent experiments ±SD. ***P < 0.01 by ANOVA analysis.

Inhibition of Braf35 sumoylation by iBraf. (A) iBraf inhibits Braf35 sumoylation. Numbers are the amount of transfected HA-iBraf–expressing vector with respect to the amount of Flag-Braf35–expressing vector. (B) The coiled-coil domain of iBraf is required for inhibition of Braf35 sumoylation. (C) The coiled-coil domain of Braf35 is required for iBraf-dependent inhibition of Braf35 sumoylation. (A–C) 293T cells were transfected with expression vectors for the indicated proteins. Flag-tagged, or HA-tagged proteins were detected by Western blot using anti-Flag or anti-HA antibodies. (Lower) Inputs of the indicated proteins. (D) Sumoylation of endogenous BRAF35 depends on the level of iBraf. 293T cells were transfected with expression vectors for His-HA-SUMO1 and Flag-iBraf as indicated. Sumoylated proteins were purified using nickel-affinity chromatography and subjected to Western blot analysis using anti-Braf35 antibodies. (Lower) The presence of Flag-iBraf in the extracts was verified by Western blot using anti-Flag antibodies.

Braf35 sumoylation is required for LSD1–CoREST repression. (A and B) Braf35 is sumoylated. 293T cells were transfected with expression vectors for the indicated proteins. wt, wild type Flag-Braf35; 4KR, Flag-Braf35-4KR mutant; dnUBC9, dominant-negative UBC9 (C93S). Flag-tagged proteins were detected by Western blot using anti-Flag antibodies. (C) Sumoylation of Braf35 is not required for its assembly in the LSD1–CoREST complex. 293T cells were transfected with expression vectors encoding Flag-Braf35 (wt), Flag-Braf35-3KR (3KR), or Flag-Braf35-4KR (4KR) sumoylation-defective mutants. Whole-cell extracts were subjected to immunoprecipitation with anti-Flag antibody (IP α-Flag) or mouse purified IgG as a control and analyzed by Western blot using the indicated antibodies. The asterisk denotes bands corresponding to immunoglobulins. (D) Derepression of SCN1A, SCN2A2, and SCN3A genes in HeLa cells. SCN1A, SCN2A2, and SCN3A mRNA levels were determined by RT-qPCR from HeLa cells transfected with expression vectors for the indicated proteins. (E) Overexpression of Braf35-4KR is coupled with dissociation of LSD1 and elevated levels of AcH3, H3K4me2, and H3K4me3 at the SCN1A promoter. HeLa cells transfected with Braf35, Braf35-4KR, or empty vector were subjected to ChIP assays with the indicated antibodies. Relative fold values indicate occupancies relative to empty vector set at 1. (D and E) Values are the average of multiple experiments (n ≥ 3) ±SD. *P < 0.1, **P < 0.05, ***P < 0.01 by ANOVA analysis, compared with cells transfected with empty vector.

iBraf forms homodimers and heterodimers with Braf35. (A and C) iBraf homodimerizes and heterodimerizes with Braf35 in vitro. One microgram of GST-iBraf or GST proteins bound to glutathione-Sepharose beads was incubated with in vitro translated ³⁵S-labeled iBraf (A) or ³⁵S-labeled Braf35 (C). Bound proteins and 20% of the input were subjected to SDS/PAGE. Gels were dried and autoradiographed. (B) iBraf homodimerizes in vivo. 293T cells were transfected with expression vectors encoding HA-iBraf or Flag-iBraf. Whole-cell extracts were subjected to immunoprecipitation with anti-HA antibodies (IP α-HA) or mouse purified IgG as a control and analyzed by Western blot using α-Flag antibodies. (D) Endogenous iBRAF and BRAF35 interact in vivo. Whole-cell extracts from 293T cells were subjected to immunoprecipitation with anti-iBraf antibody or rabbit purified IgG as a control. Immunoprecipitated proteins and 3% of the input were analyzed by Western blot with anti-Braf35 antibodies. (E–G) The coiled-coil domains of iBraf and Braf35 are required for iBraf–iBraf and iBraf–Braf35 interactions. Plasmids expressing the indicated proteins were transfected in 293T cells. Flag-iBraf (E), Flag-Braf35 (F), or HA-iBraf (G) were immunoprecipitated and analyzed by Western blot using the indicated antibodies. (H) Composition of LSD1–CoREST complexes upon overexpression of iBraf. 293T cells were transfected with expression vectors encoding HA (-), HA-iBraf (wt), or HA-iBrafΔcc (Δcc). Cell extracts were subjected to immunoprecipitation with anti-LSD1 antibodies or purified IgG as a control. Immunoprecipitated proteins and 3% of the input were analyzed by Western blot with the indicated antibodies.

iBraf antagonizes Braf35-dependent inhibition of differentiation. (A) P19 cells were transfected with expression vectors for the indicated proteins together with a GFP expression vector. Three days after transfection, cells were analyzed by TuJ1 immunostaining and GFP expression. TuJ1-positive cells were scored as a percentage of GFP-positive transfected cells. (B) Sections from HH21 chicken embryos stained with TuJ1 and DAPI 30 h postelectroporation of plasmids encoding Neurogenin2 (B2), Neurogenin2 and Braf35 (B3), and Neurogenin2, Braf35, and iBraf (B4), or an empty vector (B1) together with GFP expression vector. (Scale bars, 50 μm.) (C) Quantification of data presented in B as a percentage of TuJ1-positive cells per total number of GFP-positive cells. Data are the average of three independent experiments ±SD. **P < 0.05, ***P < 0.01, by ANOVA analysis.