anti mitf nb110 10872 antibody  (Novus Biologicals)

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: Mitf, but not GNAS, and phospho-ERK1/2 were uniformly increased in heterozygous hSASH1(Y551D) gene knock-in BABL/cJ mice compared to wild-type mice. (A) Downregulation of SASH1 was induced by the Y551D SASH1 mutation in homozygous mice of the F2 generation compared to that of wild-type mice. GNAS and Mitf expression was attenuated by the downregulation of SASH1; however, phospho-ERK1/2 expression was increased (left panel). The total densitometry values of these proteins were also compared collectively (lower right panel). Upper right panel: # P<0.01 vs. all 3 wild-type mice, ** P<0.001 vs. all 3 wild-type mice; ns, not significant vs. all 3 wild-type mice. Lower right panel: # P<0.01, homozygous mice vs. wild-type mice, ** P<0.001 vs. all 3 wild-type mice. (B) SASH1, GNAS and Mitf expression was not attenuated by the Y551D-SASH1 mutation in the F3 generation, as indicated by western blot and statistical analyses. ns: No significance vs. all four wild-type mice. Mitf, but not GNAS, and phospho-ERK1/2 were uniformly increased in heterozygous hSASH1(Y551D) gene knock-in BABL/cJ mice compared to wild-type mice. (C-E) Tail biopsies of wild-type mice and heterozygous mice of the F2 generation were lysed, ultrasonicated and subjected to western blot analysis. The results of western blot analysis indicated that the expression of SASH1, GNAS, phospho-ERK1/2 and Mitf was enhanced in the heterozygous hSASH1(Y551D) gene knock-in BABL/cJ mice of the F2 generation compared to wild-type mice. (D) The densitometry values of SASH1, GNAS, phospho-ERK1/2 and Mitf of 11 heterozygous and 3 wild-type mice were compared, and (E) the total densitometry values of these proteins were also compared collectively. (D) * P<0.05 vs. all 3 wild-type mice, # P<0.01 vs. all 3 wild-type mice, ** P<0.001 vs. all 3 wild-type mice. (E) * P<0.05 vs. wild-type mice; # P<0.01 vs. wild-type mice, ** P<0.001 vs. wild-type mice. Mitf, but not GNAS, and phospho-ERK1/2 were uniformly increased in heterozygous hSASH1(Y551D) gene knock-in BABL/cJ mice compared to wild-type mice. (F-H) Western blot analysis revealed that the expression of Mitf was enhanced in heterozygous human Y551D SASH1 gene knock-in mice of the F3 generation compared with wild-type mice. (G) The densitometry values of SASH1, GNAS, phospho-ERK1/2 and Mitf of 10 heterozygous and 4 wild-type mice were compared, and (H) the total densitometry values of these proteins were also compared collectively. (G) * P<0.05 vs. all 3 wild-type mice, # P<0.01 vs. all 3 wild-type mice; ** P<0.001 vs. all 3 wild-type mice. (H) ** P<0.001 vs. wild-type mice. For all panels, ns, not significant.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Gene Knock-In, Mutagenesis, Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: Mitf, but not GNAS, and phospho-ERK1/2 expression is increased in heterozygous human Y551D SASH1 knock-in mice compared to homozygous mice. (A) Western blot analysis indicated that SASH1 was downregulated in heterozygous human Y551D-SASH1- knock-in mice of the F2 generation compared to homozygous mice. However, the protein levels of phospho-ERK1/2 and Mitf were upregulated in heterozygous mice compared to those of homozygous mice. (B and C) The densitometry values of SASH1, GNAS, phospho-ERK1/2 and Mitf of 11 heterozygous and 2 homozygous mice of the F2 generation were compared, and the total densitometry values of these proteins were also compared collectively, respectively. (B) * P<0.05 vs. both homozygous mice, # P<0.01 vs. both homozygous mice; ** P<0.001 vs. both homozygous mice. (C) # P<0.01 vs. homozygous mice, ** P<0.001 vs. homozygous mice, * P<0.05 vs. homozygous mice. (D) Western blot analysis demonstrated that the expression of Mitf and GNAS was enhanced in heterozygous human Y551D SASH1 knock-in mice of the F3 generation compared to homozygous mice. Mitf, but not GNAS, and phospho-ERK1/2 expression is increased in heterozygous human Y551D SASH1 knock-in mice compared to homozygous mice. (E and F) The densitometry values of SASH1, GNAS, phospho-ERK1/2 and Mitf of 11 heterozygous and 1 homozygous F3 generation mice were compared. (F) The total gray values of these proteins were also compared collectively. (E) * P<0.05 vs. one homozygous mouse, respectively, # P<0.01 vs. one homozygous mouse, respectively, ** P<0.001 vs. one homozygous mouse, respectively. (F) ** P<0.001 vs. homozygous mice. (G) The densitometry values of SASH1, Mitf, GNAS and the ratio of p-ERK/ERK in 11 heterozygous mice of the F2 generation and 10 heterozygous mice of the F3 generation were compared. For all panels, ns, not significant.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Expressing, Knock-In, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: In the F2 generation, the number of Mitf- and Mitf-positive epithelial cells was enhanced in heterozygous human Y551D SASH1 knock-in mice. (A) Representative images (magnification, ×10) of Mitf in 3 wild-type mice, 2 homozygous mice and 11 heterozygous mice. (B) A total of 5 random visual fields in each section of 16 mice, including wild-type, homozygous and heterozygous mice, were photographed. The Mitf-positive epithelial cells in the tail tissues of wild-type, homozygous and heterozygous mice were counted and analyzed statistically. Representative Mitf-positive cells, which were stained dark brown in the nucleus, are indicated by red arrows. * P<0.01. (C) The staining intensity and percentage of Mitf-positive cells per mouse were calculated, scored and analyzed statistically. * P<0.05, *** P<0.001; ns, not significant.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Knock-In, Staining

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: In the F3 generation, the number of Mitf- and Mitf-positive epithelial cells was augmented in heterozygous human Y551D SASH1 knock-in mice. (A) Representative images (magnification, ×10) of Mitf in 4 wild-type mice, 1 homozygous mouse and 11 heterozygous mice. (B) A total of 51 visual fields in each section of 15 mice, including wild-type, homozygous and heterozygous mice, were photographed. The Mitf-positively stained epithelial cells in the tail tissues of wild-type, homozygous and heterozygous mice were calculated in 3 visual fields and analyzed statistically. Representative Mitf-positive cells, which were stained yellowish-brown in the nucleus, are indicated by red arrows. * P<0.01. (C) Staining intensity and percentage of Mitf-positive cells per mouse were calculated, scored and analyzed statistically. ** P<0.01 *** P<0.001; ns, not significant.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Knock-In, Staining

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: Mitf expression is promoted by mutated SASH1 in vitro and in epithelial tissues affected by the Y551D-SASH1 mutation. (A) Exogenous SASH1 is not associated with endogenous Mitf in 293T cells. GFP-SASH1 was transfected into 293T cells. At 48 h following transfection, transfected cells were lysed, GFP-SASH1 was immunoprecipitated, and the associated endogenous Mitf was analyzed by IP-WB analyses. (B) Expression of endogenous Mitf was induced by Y551D SASH1. Exogenous Y551D SASH1 and a wild-type SASH1 were introduced into 293T cells. Following transfection, transfected cells were lysed and subjected to western blot analyses. ** P<0.001 vs. wild-type SASH1 and *** P<0.001 vs. blank control. (C) In the lesional epithelial tissues of Y551D SASH1 -affected individuals, SASH1- and Mitf-positive cells were demonstrated in different epithelial layers of the affected epithelial tissues and calculated and analyzed statistically. ** P<0.01, *** P<0.001. Upregulation of SASH1 and enhanced Mitf were also induced in the affected tissues. Magnification, ×40. Mitf-positive cells, which were stained dark brown in the nucleus, are indicated by red arrows. (D) More melanin was synthesized and present in different epithelial layers of the affected skin epithelial tissues. Magnification, ×40.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Expressing, In Vitro, Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Control, Staining, Synthesized