Journal: Virulence

Article Title: Streptococcus suis subtilisin-like serine proteases SspA-1 and SspA-2 interplay with complement C3a and C5a to facilitate bacterial immune evasion and infection.

doi: 10.1080/21505594.2023.2301246

Figure Lengend Snippet: Figure 4. S. suis strains at stationary phase (4 × 106 CFU) were incubated with GST_C3a_GFP (0.5 μg) or GST_C5a_GFP (0.5 μg) in TSB for 6, 12 and 16 hours. Protease inhibitor cocktail in GST_C3a_GFP or GST_C5a_GFP without S. suis strains was used as a negative control. Monoclonal antibodies specific for C3 or C5 were applied for C3a and C5a immunodetection, respectively.

Article Snippet: Mouse anti-C5 monoclonal antibody (Proteintech, Wuhan, China) was applied at 37 °C for 1 hour to test the C5a binding capacity.

Techniques: Incubation, Protease Inhibitor, Negative Control, Bioprocessing, Immunodetection

Journal: International journal of cancer

Article Title: A novel NF-kappaB inhibitor DHMEQ selectively targets constitutive NF-kappaB activity and induces apoptosis of multiple myeloma cells in vitro and in vivo.

doi: 10.1002/ijc.20688

Figure Lengend Snippet: FIGURE 5 – Inhibition of constitutive NF-B binding activity by DHMEQ in primary MM cells. (a) Primary MM cells were treated with or without 10 g/ml of DHMEQ for 16 hr. Nuclear extracts were examined by EMSA using a radiolabeled NF-B specific probe. Positions of shifted bands are indicated on the left (left panel). Nuclear extracts from untreated primary MM cells were subjected to supershift analysis with antibodies specific for p50, p65, p52, c-Rel and RelB. Antibodies used are indicated on the top (right panel). M, MM samples. (b) Subcomponents of NF-B constitutively activated in PBMC with or without DHMEQ treatment were determined by supershift analysis. PBMC were treated with or without 10 g/ml of DHMEQ for 16 hr. Nuclear extracts were examined for NF-B binding activity by EMSA with a radiolabeled NF-B specific probe. Nuclear extracts of untreated PBMC were subjected to supershift analysis with antibodies specific for p50, p65, p52, c-Rel and RelB. Antibodies used are indicated on the top. (c) DHMEQ reduces cell viability of primary MM cells. Primary MM cells and PBMC were treated with 10 g/ml of DHMEQ for 48 hr. Cell viability was measured by MTT assay. The relative viabilities of treated samples are expressed as percentages of those of untreated ones. Data represent the mean SD percentage of the relative viability of 3 independent experiments. C, PBMC sample. *, more significant than controls (p 0.01). (d) Nuclear fragmentation induced by treatment with DHMEQ in primary MM cells. Primary MM cells and PBMC were treated with 10 g/ml of DHMEQ for 24 hr and subjected to staining with Hoechst 33342.

Article Snippet: Antibodies used for supershift assays were as follows: anti-p50 (C-19) goat polyclonal antibody, anti-p65 (C-20) rabbit polyclonal antibody and mouse monoclonal antibodies for c-Rel (B-6), p52 (C-5) and RelB (all from Santa Cruz).

Techniques: Inhibition, Binding Assay, Activity Assay, MTT Assay, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling

doi: 10.1007/s00018-025-05694-w

Figure Lengend Snippet:

Article Snippet: Mouse anti-AP2α1 (α-adaptin 1) (C-5) , Santa Cruz Biotechnology , Cat#sc-398024.

Techniques: Marker, Recombinant, Lysis, Blocking Assay, Protease Inhibitor, Transfection, Bicinchoninic Acid Protein Assay, Derivative Assay, Plasmid Preparation, Software, Imaging

Journal: Computational and structural biotechnology journal

Article Title: Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell.

doi: 10.1016/j.csbj.2023.03.015

Figure Lengend Snippet: Fig. 2. Inhibition of ULK1 results in a decrease of pICln phosphorylation in vivo A, Colocalization of ULK1 and pICln phospho and total. Cells were fixed with 4% PFA and afterwards permeabilized with Triton X-100 to visualize ULK1 (red) and pICln total or phosphospecific pICln (green). The DNA was stained with DAPI (blue), scale bars 10 µm. B, HEK293T cells were treated with 30 µM ULK inhibitor MRT67307 for 5 h or transfected with 50 nM Non-targeting control pool (NT) control or ULK1 siRNA for 72 h. Cells were fixed as described in A to visualize ULK1 (red) and pICln total (green). The DNA was stained with DAPI (blue), scale bars 10 µm. C, HEK293T cells were treated as described in B to visualize phosphospecific pICln (red) and pICln total (green). The DNA was stained with DAPI (blue), scale bars 10 µm. D, Inhibition or knockdown of ULK1 decreases pICln phosphorylation in vivo. Quantification of the intensity ratio between phosphospecific pICln and pICln total was made in Fiji and the diagram in Origin, the values for MRT67307 treatment are normalized on HEK wt value, and the values for ULK1 siRNA are normalized on NT control. The significance of the values was analyzed using a Mann-Whitney U test, the p-value of HEK wt vs. MRT67307 and NT control vs. ULK1 siRNA was below 0.005, indicating significant differences between the values.

Article Snippet: To control whether ULK1 siRNA worked and if the inhibition or knockdown of ULK1 total has an effect on pICln total localization the following antibodies were used: ULK1 Prestige Antibody Sigma (HPA063990) rabbit host and pICln C5 antibody Santa Cruz mouse host (sc-130668).

Techniques: Inhibition, Phospho-proteomics, In Vivo, Staining, Transfection, Control, Knockdown, MANN-WHITNEY

Journal: Computational and structural biotechnology journal

Article Title: Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell.

doi: 10.1016/j.csbj.2023.03.015

Figure Lengend Snippet: Fig. 4. ULK1 phosphorylation of pICln increases UsnRNP biogenesis and spliceosomal activity A, The HIV-1-based splicing reporter contains a test 5‘splice site (5‘ss) with a mutated version of viral 5′ss D4 termed „− 1G3U“. This splice site carries two nucleotide substitutions: A-to-G at position − 1 (−1 G) and A-to-U at position + 3 (+3 U) and is poorly recognized by the endogenous U1 snRNA due to a mismatch at position + 3. However, splice site recognition is efficiently restored following the co-expression of a U1 snRNA with a compensatory U-to-A nucleotide exchange at position + 6, indicating that the modified U1 is assembled into functional snRNP particles within the cell. Uppercase letters within the splice site sequences represent complementary residues, while lowercase letters represent mismatches to the U1. Mutations are highlighted in red. Base-pairing at position + 3 is highlighted by a light red box and increased font size. B; C, HEK293T cells were treated with 30 µM inhibitor MRT67307 and transiently transfected with different splicing reporter constructs (see methods section for more details). After 20 h of inhibitor treatment cells were harvested to perform RNA isolation. Quantitative RT-PCR was executed and mRNA was analyzed by assessing the respective ratio of spliced/unspliced form, the p value was below 0.005 (B). The equal averages including differences between inhibitor- treated and untreated spliced and unspliced forms are listed in C (n = 3).

Article Snippet: To control whether ULK1 siRNA worked and if the inhibition or knockdown of ULK1 total has an effect on pICln total localization the following antibodies were used: ULK1 Prestige Antibody Sigma (HPA063990) rabbit host and pICln C5 antibody Santa Cruz mouse host (sc-130668).

Techniques: Phospho-proteomics, Activity Assay, Expressing, Modification, Functional Assay, Transfection, Construct, Isolation, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

doi: 10.1038/s41598-024-53651-7

Figure Lengend Snippet: The co-expression of C5AR1 and its ligand C5a in intracranial aneurysm lesions. Specimens of intracranial aneurysm (IA) lesions (Aneurysm) or control arterial walls (Superficial temporal artery in ( A ), Anterior cerebral artery-olfactory artery bifurcation in ( B )) from human cases ( A ) or rats ( B ) were harvested and examined in immunohistochemistry. The representative images of immunofluorescent staining of IA lesions for C5AR1 (green), C5a/C5a des-arg (red in ( A )), C5 (red in ( B )), and merged images with nuclear staining by DAPI (blue) are shown. A demagnified image of lower panels in ( B ) is shown on the right. The square in the demagnified image corresponds to the area shown in the magnified images. Scale bar: 50 μm.

Article Snippet: Primary antibodies used were as follows; rabbit polyclonal anti-CD88 (C5AR1) antibody (#PAB26140, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD31 antibody (clone JC70A, #M0823, Dako, Glostrup, Denmark), mouse monoclonal anti-smooth muscle α-actin antibody (clone 1A4, #14-9760-82, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-myeloperoxidase antibody (#ab9535, abcam, Cambridge, UK), mouse monoclonal anti-human C5a/C5a des-Arg antibody (clone 2952, #HM2079, Hycult Biotech, Wayne, PA, USA), rabbit polyclonal anti-tPA antibody (#10147-1-AP, Proteintech Group, Rosemont, IL, USA), and rabbit polyclonal anti-plasminogen antibody (#GTX102877, Gene Tex, Irvine, CA, USA).

Techniques: Expressing, Control, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

doi: 10.1038/s41598-024-53651-7

Figure Lengend Snippet: Tissue-type Plasminogen Activator as an up-stream factor to produce C5a from C5. ( A ) The expression of tissue-type Plasminogen Activator (tPA) and Plasminogen in intracranial aneurysm (IA) lesions. IA lesions from human cases or induced in rats were harvested subjected to immunohistochemical analyses. The images of immunofluorescent staining of IA lesions for tPA (green), Plasminogen (red), and merged images with nuclear staining by DAPI (blue) are shown. Scale bar: 50 μm. ( B ) Co-expression of tPA with C5a in IA lesions. Human IA lesions were harvested subjected to immunohistochemical analyses. The images of immunofluorescent staining of IA lesions for C5a/C5a des-arg which is the derivative of C5a (green), tPA (red), and merged images with nuclear staining by DAPI (blue) are shown. Scale bar: 50 μm. ( C ) Enzymatic cleavage of C5 into C5a by Plasmin. Recombinant C5 (100 μg/ml) was co-incubated with each dose of recombinant Plasmin as indicated (0–100 μg/ml) for 1.5 h in a cell-free system. The incubation mixture was subjected to western blot analyses. The representative images from the western blot analyses to detect C5 or C5a are shown. M; size marker. ( D ) The alternation in the coagulation-fibrinolysis system in IA lesions of rats. The heat map of genes related with the coagulation-fibrinolysis system from the comprehensive gene expression profile data set (GEO accession: GSE161044) is shown.

Article Snippet: Primary antibodies used were as follows; rabbit polyclonal anti-CD88 (C5AR1) antibody (#PAB26140, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD31 antibody (clone JC70A, #M0823, Dako, Glostrup, Denmark), mouse monoclonal anti-smooth muscle α-actin antibody (clone 1A4, #14-9760-82, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-myeloperoxidase antibody (#ab9535, abcam, Cambridge, UK), mouse monoclonal anti-human C5a/C5a des-Arg antibody (clone 2952, #HM2079, Hycult Biotech, Wayne, PA, USA), rabbit polyclonal anti-tPA antibody (#10147-1-AP, Proteintech Group, Rosemont, IL, USA), and rabbit polyclonal anti-plasminogen antibody (#GTX102877, Gene Tex, Irvine, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Recombinant, Incubation, Western Blot, Marker, Coagulation

Journal: Scientific Reports

Article Title: C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

doi: 10.1038/s41598-024-53651-7

Figure Lengend Snippet: C5a–C5AR1 signaling cascade-dependent induction of pro-inflammatory factors. ( A – C ) The C5a–C5AR1 signaling cascade-dependent induction of pro-inflammatory genes in phorbol myristate acetate-primed cell line from human case with diffuse histiocytic lymphoma, U937 cell line. Cells were treated with recombinant C5a as indicated in the panels (0–10 h in ( A ), 0–100 nM in ( B ), 10 nM and 10 h in ( C ). Some cells were pre-treated with the selective C5AR1 inhibitor, W54011 (300 nM, 30 min) prior to the treatment with recombinant C5a. Total RNA was then purified from treated cells subjected to quantitative RT-PCR analyses to examine the induction of TNF (a gene for TNF-α), IL1B or PTGS2 (a gene for COX-2; cyclooxygenase-2). Data represents as mean ± s.e.m (( A ), n = 4; ( B , C ), n = 6). Statistical analysis was done by the Wilcoxon rank sum test in ( A ), the Kruskal–Wallis test followed by the Steel–Dwass test in ( B , C ). *p < 0.05. ( D ) Co-expression of C5a with pro-inflammatory factors in intracranial aneurysm (IA) lesions. IA lesions induced in rats were harvested subjected to immunohistochemical analyses. The images of immunofluorescent staining of IA lesions for C5ar1 (green), TNF-α (red), IL-1β (red), COX-2 (red), and merged images with nuclear staining by DAPI (blue) are shown. Scale bar: 50 μm.

Article Snippet: Primary antibodies used were as follows; rabbit polyclonal anti-CD88 (C5AR1) antibody (#PAB26140, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD31 antibody (clone JC70A, #M0823, Dako, Glostrup, Denmark), mouse monoclonal anti-smooth muscle α-actin antibody (clone 1A4, #14-9760-82, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-myeloperoxidase antibody (#ab9535, abcam, Cambridge, UK), mouse monoclonal anti-human C5a/C5a des-Arg antibody (clone 2952, #HM2079, Hycult Biotech, Wayne, PA, USA), rabbit polyclonal anti-tPA antibody (#10147-1-AP, Proteintech Group, Rosemont, IL, USA), and rabbit polyclonal anti-plasminogen antibody (#GTX102877, Gene Tex, Irvine, CA, USA).

Techniques: Recombinant, Purification, Quantitative RT-PCR, Expressing, Immunohistochemical staining, 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, 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

Journal: bioRxiv

Article Title: β -actin is essential for structural integrity and physiological function of the retina

doi: 10.1101/2023.03.27.534392

Figure Lengend Snippet: Differential interaction of cytoplasmic actins with actin associated proteins. (A) Different interaction with β - and γ -actin was shown as the relative enrichment of a protein in phalloidin-pulldown of the lysates of wildtype and Actb cg/cg retina. The p-values are from and . (B) The immunostaining of myosin-VIIa indicated the increased interaction of myosin-VIIa with γ actin (C). Scale bar: 5 µ m. The p-values are from paired Student’s t-test with n =4 pairs. Myh9/Myo7a: gene symbol of myosin-IIa/myosin-VIIa protein. For other gene symbols in (A), and show the protein names.

Article Snippet: The following primary antibodies were used: mouse anti- β -actin (1:100 dilution, clone 4C2, MABT825, Millipore-Sigma), mouse anti- γ -actin (1:100 dilution, clone 2A3, MABT824, Millipore-Sigma), mouse anti-Ezrin (1:100 dilution, CPTC-ezrin-1, AB 2100318, Developmental Studies Hybridoma Bank, a gift from Dr. A. Bretscher, Cornell University), rabbit anti-p-ERM (1:100 dilution, a gift from Dr. A. Bretscher, Cornell University [ ]), mouse anti-Myosin VIIa (1:50 dilution, clone C-5, sc-74516, Santa Cruz Biotechnology Inc.), rat anti-HCAM (CD44) (1:10 dilution, clone IM7, sc-18849, Santa Cruz Biotechnology Inc.), rabbit anti-G β 1 (1:100 dilution, clone, C-16, sc-379, Santa Cruz Biotechnology Inc.), mouse anti-rhodopsin (1:100 dilution, clone 1D4, sc-57432, Santa Cruz Biotechnology Inc.).

Techniques: Immunostaining