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Figure 2
Anti-Brd2b peptide antibody detects major protein products in zebrafish embryos and oocytes, that are reduced in brd2bMO morphants. (A,B) Western blot (A) of total ovary (ov) and staged embryos (4 h, 24 h, 48 h) probed with anti-Brd2b peptide antibody. Blot was prestained (B) to assess total protein and relative loading across lanes. Total ovary samples often show a large amount of high molecular weight staining corresponding to yolk proteins that make up predominant products in this tissue; nevertheless, these do not interfere with the detection of Brd2b. A product over 150 kD is consistently detected in ovaries and 48 hpf embryos and sometimes faintly in 4 and 24 hpf embryos (top arrowheads in A); a product between 50 and 75 kD is also consistently detected in 48 hpf embryos (bottom arrowhead) and sometimes also in 4 hpf and 24 hpf embryos (see Supplementary Figure S1). (C,D) Duplicate blot of A probed with anti-Brd2b peptide antibody in the presence of peptide antigen for peptide competition assay (C). The blot was prestained (D) to assess total protein and relative loading across lanes before antibody + peptide incubation. Peptide competition prevents all antibody signals on blot (C), supporting specificity of antibody for Brd2b epitope. Prestaining of blot using Memcode protein stain (D) assures that lack of anti-Brd2b signal in C is due to peptide competition rather than lack of protein in lanes. (E) Immunohistochemical analysis of staged oocytes (I?IV) and higher magnification stage IV oocyte (IV) using anti-Brd2b peptide antibodies, and showing eventual localization to the micropyle and future animal pole. (F,G) Western blot to assess efficacy of brd2bMO morpholino knockdown. Twenty-four hours post-fertilization embryo treatment groups: uninjected (wt), control 5-base mismatch morpholino-injected (mis5), and brd2bMO1-injected (2bMO), probed with anti-Brd2b peptide antibody (F). The blot was prestained (G) to assess total loading and relative loading across lanes. When relative loading is taken into account, there is a 3-fold reduction in Brd2b (~150 kD band) in brd2bMO-treated embryos (2ng total) as measured by ImageJ (FiJI) using band pixel densities (see Methods). Anti-Brd2B peptide antibody used in all immunoblot experiments in this study was raised against an antigenic epitope between NLS and ET (see Materials and Methods), and detects only the long isoform (Brd2b-L). Bottom edges of Western blots are indicated by horizontal line in (A, C and F). mw = Precision plus dual color molecular weight standard (BioRad); ch = Precision plus Western Chemiluminescent marker (BioRad).