Production and Characterization of Monoclonal Antibodies against Human Nuclear Protein FAM76B

<div><p>Human FAM76B (hFAM76B) is a 39 kDa protein that contains homopolymeric histidine tracts, a targeting signal for nuclear speckles. FAM76B is highly conserved among different species, suggesting that it may play an important physiological role in normal cellular functions. However, a lack of appropriate tools has hampered study of this potentially important protein. To facilitate research into the biological function(s) of FAM76B, murine monoclonal antibodies (MAbs) against hFAM76B were generated by using purified, prokaryotically expressed hFAM76B protein. Six strains of MAbs specific for hFAM76B were obtained and characterized. The specificity of MAbs was validated by using FAM76B^-/- HEK 293 cell line. Double immunofluorescence followed by laser confocal microscopy confirmed the nuclear speckle localization of hFAM76B, and the specific domains recognized by different MAbs were further elucidated by Western blot. Due to the high conservation of protein sequences between mouse and human FAM76B, MAbs against hFAM76B were shown to react with mouse FAM76B (mFAM76B) specifically. Lastly, FAM76B was found to be expressed in the normal tissues of most human organs, though to different extents. The MAbs produced in this study should provide a useful tool for investigating the biological function(s) of FAM76B.</p></div

Domain mapping for anti-hFAM76B MAbs.

<p>(A) Cartoon of the position of the six different fragments in the FAM76B gene. (B) Western blot analysis of the binding of different anti-hFAM76B MAbs to full-length and six truncated mutants of hFAM76B under denaturing conditions. (C) Map of the domains recognized by different anti-hFAM76B MABs.</p

Exogenous and endogenous mFAM76B recognized by anti-hFAM76B MAbs.

<p>(A) Intranuclear localization of overexpressed mFAM76B in HEK 293 cells revealed by immunofluorescence staining. HEK 293 cells were transfected with plasmid expressing mFAM76B-Flag and stained with anti-hFAM76B MAbs and anti-Flag MAb. Normal mouse serum was used as a negative control. TRITC-conjugated goat anti-mouse IgG was used as the secondary antibody. Nuclei were labeled with DAPI (blue). (B) Endogenous mFAM76B revealed by immunohistochemical staining with MAbs against hFAM76B. NIH/3T3 and Hepa1-6 cells were fixed with 4% paraformaldehyde and then stained with the anti-hFAM76B MAbs. Normal mouse serum was used as a negative control. Primary Abs were detected by a Vectastain ABC Kit with DAB as a substrate. Nuclei were labeled with hematoxylin. Bar = 50 μm.</p

Western blotting and immunoprecipitation with MAbs against hFAM76B.

<p>(A) All MAbs except for No. 4 recognized hFAM76B-His recombinant protein expressed in <i>E</i>. <i>coli</i>. Truncated CD133-His fusion protein was used as the negative control protein and anti-His MAb was used as the positive control antibody. (B) Recombinant full-length hFAM76B from HEK 293 cells transfected with eukaryotic expressing vector carrying hFAM76B cDNA recognized by anti-FAM76B MAbs. 1and 2 indicated the samples from two independent transfection. (C) Endogenous FAM76B expressed in HepG2 or Shsy5y cells was detected by MAbs against hFAM76B; anti-GAPDH MAb was used as the positive control antibody. (D) Loss of FAM76B expression in FAM76B^-/- HEK 293 cells revealed by MAbs against hFAM76B; anti-GAPDH MAb was used for loading control. (E) The cell lysates of HEK 293 cells overexpressing hFAM76B-Flag were subjected to immunoprecipitation with anti-FAM76B MAbs followed by immunoblotting with anti-Flag MAb. Anti-Flag antibody was used as a positive control and rabbit anti-mouse IgG was used as a negative control.</p

FAM76B expression in normal human organs.

<p>(A) brain cortex; (B) brain subcortical white matter; (C) liver; (D) spleen; (E) lung; (F) kidney; (G) adrenal gland; (H) heart. All tissues were stained with MAb No.2. FAM76B was found in most organs, though to different extents. The staining was strongest in the nuclei of lymphocytes in the spleen (D), renal tubular epithelium (F), bile duct (C) and glial cells in the brain (A, B). Intermediate staining was observed in the hepatocytes (C), lung (E), and neurons (A). Glomeruli (F) and cardiac muscle (H) were overall minimally stained. Normal mouse serum was used as a negative control. Primary Abs were detected by a Vectastain ABC Kit with DAB as a substrate. Nuclei were labeled with hematoxylin. Bar = 100 μm.</p

Mouse FAM76B recognized by MAbs against hFAM76B.

<p>(A) FAM76B human and mouse protein sequences were aligned using the Blast 2 Sequences program. Asterisks indicate different amino acids between these two sequences. (B) Exogenous overexpression of mFAM76B in HEK 293 cells was detected by Western blot with MAbs against hFAM76B. All MAbs except No. 4 reacted with the overexpressed mFAM76B sensitively and specifically. (C) Endogenous mFAM76B in NIH/3T3 and Hepa1-6 cells were revealed by Western blot using MAb No.1, 2 and 5.</p

Immunocytochemical detection of endogenous hFAM76B.

<p>HepG2 and Shsy5y FAM76B^-/- HEK 293 cells were fixed with 4% paraformaldehyde and then stained with anti-FAM76B MAbs. Normal mouse serum was used as a negative control. Primary Abs were detected by a Vectastain ABC Kit with DAB as a substrate. Nuclei were labeled with hematoxylin. Scale bar = 50μm.</p

Intranuclear distribution of exogenous hFAM76B protein.

<p>(A) Immunofluorescent staining with anti-hFAM76B MAbs on HEK 293 cells overexpressing hFAM76B. HEK 293 cells were transfected with hFAM76B-Flag-expressing plasmid. Twenty-four hours after transfection, the cells were fixed with 4% paraformaldehyde and then stained with the anti-hFAM76B MAbs and an anti-Flag MAb. Normal mouse serum was used as a negative control. FAM76B^-/- HEK 293 cells were used for the negative control. TRITC-conjugated goat anti-mouse IgG was used as the secondary antibody. Nuclei were labeled with DAPI (blue). (B) Confocal study of hFAM76B and SC35, an endogenous marker of nuclear speckle compartments. HEK 293 cells were transfected with vector expressing hFAM76B and immunostained with anti-hFAM76B MAb No. 2 (red) and rabbit anti-SC-35 antibody (green). Scale bar = 10 μm.</p

Rescue the Failed Half-ZFN by a Sensitive Mammalian Cell-Based Luciferase Reporter System

<div><p>ZFN technology is a powerful research tool and has been used for genome editing in cells lines, animals and plants. The generation of functional ZFNs for particular targets in mammalian genome is still challenging for an average research group. The modular-assembly method is relatively fast, easy-to-practice but has a high failure rate. Some recent studies suggested that a ZFP with low binding activity might be able to form a working ZFN pair with another binding active half-ZFP. In order to unveil the potential ZFP candidates among those with low binding activities, this paper established a highly sensitive mammalian cell-based transcriptional reporter system to assess the DNA binding activities of ZFPs by inserting multiple copies of ZFN target sequence fragment (TSF) of an interested gene (e. g., hPGRN or hVEGF). Our results showed that this system increased the screening sensitivity up to 50-fold and markedly amplified the differences in the binding activities between different ZFPs. We also found that the targeted chromosomal gene repair efficiency of each hPGRN or hVEGF ZFN pair was in proportion with the combination of the binding activities of the ZFL (Left zinc finger) and ZFR (Right zinc finger). A hPGRN ZFR with low binding ability was able to form a biological active ZFN if combined with a hPGRN ZFL with relatively high binding ability. Lastly, site-specific genome editing by hPGRN ZFNs generated by this system was confirmed by sequencing, and the PGRN knock-out cell line showed significantly decreased cell growth compared with the control. Our system will provide a valuable tool for further optimizing the nucleases with regard to specificity and cytotoxicity.</p> </div

The target sequences, corresponding key amino acid sequences and binding affinities of hVEGF2 zinc fingers (ZF).

<p>Binding affinities are expressed as mean fold-activation of transcription in the EZSS system.</p