anti mitf Search Results


mitf  (R&D Systems)
94
R&D Systems mitf
Mitf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+mitf/Human+MITF+Antibody/pmc06481345-161-75-80
Average 94 stars, based on 1 article reviews
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mitf  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology mitf
FIGURE <t>1.</t> <t>FUS</t> is in a complex with <t>MITF.</t> A, Coomassie-stained FLAG-immunoprecipitated samples run on an 8.5% SDS-polyacrylamide gel; lane 1 (mock), FLAG-immunoprecipitated lysates from parental RAW264.7 C4 cell line; lane 2 (FM), FLAG immunoprecipitated lysates from RAW264.7 C4 cell line stably expressing FLAG-MITF. Lane 3 (M) marker, FUS and MITF based on size are indicated on the right. B, pulldown of RAW264.7 C4 cell lysates using GSH-Sepharose beads incubated either with GST (lane 1) or GST-MITF (lane 2) and probed with anti-FUS antibody. Input controls for RAW264.7 C4 lysates and GST beads are depicted in middle and bottom panels, respectively. C, top panel, schematic diagram of full-length and N- and C-terminal FUS constructs used for MITF interaction mapping. Ser-, Tyr-, Gly-, and Gln-rich N-terminal activation domain (AD) and C-terminal RGG-rich RNA binding domains (RBD) are shown. Bottom panel, coimmunoprecipitation studies in COS-7 cell lysates using full-length FLAG-MITF (lanes 2–4), full-length V5-FUS (lane 1 and 2), V5-tagged N-terminal FUS (lane 3, N; aa 1–259) or V5-tagged C-terminal FUS (lane 4, C; aa 260–518) immunoprecipitated (IP) with anti-FLAG antibody and probed with anti-V5 antibody. Input controls for V5-FUS and FLAG-MITF are shown. D, top panel, schematic diagram of full-length and N- and C-terminal MITF constructs used for FUS interactionmapping.N-terminalactivationdomains(AD1andAD2denotedasAD),basic(B),helix-loop-helix(HLH),andleucinezipper(LZ)domainsareshown. Ser-73 ERK phosphorylation site and Ser-307 p38 MAPK phosphorylation site as well as Lys-182 and Lys-316 sumoylation sites are also denoted. Bottom panel, immunoprecipitates pulled with FLAG antibody from COS-7 cell lysates expressing full-length FLAG-MITF (lane 1 and 3), full-length V5-FUS (lanes 2–5), FLAG-tagged N-terminal (aa 1–178) and C-terminal (aa 178–419) MITF (lanes 4 and 5, respectively) and immunoblotted (IB) with anti-V5 antibody. Loading controls for V5-FUS and FLAG-MITF are shown at the bottom.
Mitf, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+mitf/MITF+Antibody/10__1074_slash_jbc__m113__493874-58-26-42
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mitf  (Novus Biologicals)
90
Novus Biologicals mitf
Fig. 6 Enforced KDM5B expression facilitates melanocytic lineage-directed elimination by TMECG. a Quantitation of mRNA after 24 h, 48 h, 72 h and 7 days of Cpd1 treatment of MaMel63a cells as assessed by qPCR. Mean ± SD. Shown is one representative example. b Regulation of differentiation, cytokinesis, and mitotic spindle assembly genes as detected by cDNA microarray analysis after KDM5B shRNA knockdown in WM3734 cells (n = 1). c, d Immunoblotting of melanocytic lineage and (de-)differentiation markers after 24 h of KDM5B induction in WM3734Tet3G-KDM5B cells (c) and after 72 h of Cpd1 treatment in MaMel63a cells (d). Shown are representative data (n = 2). <t>e</t> <t>Anti-MITF</t> immunostaining (upper panel) and Fontana-Masson staining (lower panels) of CM melanoma tumor grafts from Cpd1-treated vs. control mice. f MTT cell viability assay of WM3734 cells. Representative example is shown left (mean ± SD, n = 2) and corresponding IC50 values on the right. TMECG was either concurrently given together with Cpd1 (“con”) or added 3 days after Cpd1 pre-treatment (“pre”). Readout was performed after 72 h of TMECG treatment. g Persister-state-directed therapy model in vivo. Left: schematic representation of treatment dosing and timing in immunodeficient NMRI-(nu/nu)-nude mice. Right: tumor volumes of WM3734 xenografts (endpoint at day 30). TMECG was either concurrently given together with Cpd1 (“con”) or added one week after Cpd1 pre-treatment (“pre”). Mean ±SEM (6 mice in TMECG and Cpd1 control group, five mice in “con” and seven mice in “pre” group). Significance was determined by two-sided Mann–Whitney test. Source data are provided as a Source Data file.
Mitf, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+mitf/MITF+Antibody+(MITF%2F2987R)+%5BmFluor+Violet+500+SE%5D/pm35650266-373-35-53
Average 90 stars, based on 1 article reviews
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monospecific antibody  (Atlas Antibodies)
93
Atlas Antibodies monospecific antibody
Fig. 6 Enforced KDM5B expression facilitates melanocytic lineage-directed elimination by TMECG. a Quantitation of mRNA after 24 h, 48 h, 72 h and 7 days of Cpd1 treatment of MaMel63a cells as assessed by qPCR. Mean ± SD. Shown is one representative example. b Regulation of differentiation, cytokinesis, and mitotic spindle assembly genes as detected by cDNA microarray analysis after KDM5B shRNA knockdown in WM3734 cells (n = 1). c, d Immunoblotting of melanocytic lineage and (de-)differentiation markers after 24 h of KDM5B induction in WM3734Tet3G-KDM5B cells (c) and after 72 h of Cpd1 treatment in MaMel63a cells (d). Shown are representative data (n = 2). <t>e</t> <t>Anti-MITF</t> immunostaining (upper panel) and Fontana-Masson staining (lower panels) of CM melanoma tumor grafts from Cpd1-treated vs. control mice. f MTT cell viability assay of WM3734 cells. Representative example is shown left (mean ± SD, n = 2) and corresponding IC50 values on the right. TMECG was either concurrently given together with Cpd1 (“con”) or added 3 days after Cpd1 pre-treatment (“pre”). Readout was performed after 72 h of TMECG treatment. g Persister-state-directed therapy model in vivo. Left: schematic representation of treatment dosing and timing in immunodeficient NMRI-(nu/nu)-nude mice. Right: tumor volumes of WM3734 xenografts (endpoint at day 30). TMECG was either concurrently given together with Cpd1 (“con”) or added one week after Cpd1 pre-treatment (“pre”). Mean ±SEM (6 mice in TMECG and Cpd1 control group, five mice in “con” and seven mice in “pre” group). Significance was determined by two-sided Mann–Whitney test. Source data are provided as a Source Data file.
Monospecific Antibody, supplied by Atlas Antibodies, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+mitf/Anti-MITF/10__4172_slash_2155___9929__1000129-63-1-6
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mitf  (Proteintech)
95
Proteintech mitf
(A) Immunofluorescence images of PIG1 cells transfected <t>with</t> <t>MC1R-mEmerald</t> (green) and mock-treated, or treated with UV, 100 nM α-MSH, or both. Cells were treated with α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). Scale bar, 5 μm. (B) MC1R ciliary enrichment analysis of panel A as previously reported . Ciliary and cell body intensity of MC1R was measured using ImageJ. Enrichment at the cilium is expressed as: (integrated density at the cilium)/(integrated density in the cell body). Enrichment > 1 indicates higher localization of mEmerald tagged MC1R at the primary cilium than at the cell body. Every replicate was represented as a superplot. ( n = 30 ciliated cells from 3 different replicates) (C, D) Immunofluorescence images (C) of A375 MC1R-KO cells transfected with WT, R151C, or R160W MC1R-mEmerald (green) and mock-treated or treated with 100 nM α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). The percentage of ciliated cells with ciliary MC1R localization was quantified in panel D ( n = 3 independent experiments). Scale bar, 5 μm. (E) Immunofluorescence images of MC1R-mEmerald transfected primary human melanocytes treated with UV/α-MSH (100 nM). Cells were treated with α-MSH for 36 h in the presence of serum after UV exposure. Cells were stained with the Arl13b (red) and TYRP1 (magenta) antibodies. Nuclei were stained with DAPI (blue). Representative images for non-ciliated cells, ciliated cells without ciliary MC1R (MC1R − cilium), and ciliated cells with ciliary MC1R (MC1R + cilium) were selected from the same slides. Scale bar, 10 μm. (F) Quantification of the number of TYRP1 puncta ( n = 20 cells) as described in E. (G) Tyrosinase activity of primary human melanocytes that were mock treated, treated with 100 nM α-MSH, 100 nM α-MSH/30 μm ciliobrevin A (Cilio A), or 100 nM α-MSH/2 mM CH for 36 h in the absence of serum after UV exposure ( n = 3 independent experiments). (H) Quantitative RT-PCR analysis of melanogenesis-related genes in primary human melanocytes treated as described in G ( n = 3 independent experiments). TYR, tyrosinase; DCT, dopachrome tautomerase; <t>MITF,</t> melanocyte inducing transcription factor; TYRP1, tyrosinase related protein 1; PMEL, premelanosome protein. (I) Immunoblot analysis of melanogenesis-related proteins in primary human melanocytes treated as described in G. GAPDH served as a control. (J) Tyrosinase activity of A375 MC1R-KO cells rescued with different forms of MC1R ( n = 5 independent experiments). Cilio-MC1R was constructed by fusing the ciliary protein Arl13b with WT, R151C, or R160W MC1R. Cells were treated with or without 100 nM α-MSH for 36 h in the absence of serum and UV. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; CH, chloral hydrate; MC1R, melanocortin 1 receptor; UV, ultraviolet.
Mitf, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+mitf/MITF+Antibody/pmc11637432-196-70-71
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rabbit polyclonal mitf antibody  (Cusabio)
90
Cusabio rabbit polyclonal mitf antibody
(A) Immunofluorescence images of PIG1 cells transfected <t>with</t> <t>MC1R-mEmerald</t> (green) and mock-treated, or treated with UV, 100 nM α-MSH, or both. Cells were treated with α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). Scale bar, 5 μm. (B) MC1R ciliary enrichment analysis of panel A as previously reported . Ciliary and cell body intensity of MC1R was measured using ImageJ. Enrichment at the cilium is expressed as: (integrated density at the cilium)/(integrated density in the cell body). Enrichment > 1 indicates higher localization of mEmerald tagged MC1R at the primary cilium than at the cell body. Every replicate was represented as a superplot. ( n = 30 ciliated cells from 3 different replicates) (C, D) Immunofluorescence images (C) of A375 MC1R-KO cells transfected with WT, R151C, or R160W MC1R-mEmerald (green) and mock-treated or treated with 100 nM α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). The percentage of ciliated cells with ciliary MC1R localization was quantified in panel D ( n = 3 independent experiments). Scale bar, 5 μm. (E) Immunofluorescence images of MC1R-mEmerald transfected primary human melanocytes treated with UV/α-MSH (100 nM). Cells were treated with α-MSH for 36 h in the presence of serum after UV exposure. Cells were stained with the Arl13b (red) and TYRP1 (magenta) antibodies. Nuclei were stained with DAPI (blue). Representative images for non-ciliated cells, ciliated cells without ciliary MC1R (MC1R − cilium), and ciliated cells with ciliary MC1R (MC1R + cilium) were selected from the same slides. Scale bar, 10 μm. (F) Quantification of the number of TYRP1 puncta ( n = 20 cells) as described in E. (G) Tyrosinase activity of primary human melanocytes that were mock treated, treated with 100 nM α-MSH, 100 nM α-MSH/30 μm ciliobrevin A (Cilio A), or 100 nM α-MSH/2 mM CH for 36 h in the absence of serum after UV exposure ( n = 3 independent experiments). (H) Quantitative RT-PCR analysis of melanogenesis-related genes in primary human melanocytes treated as described in G ( n = 3 independent experiments). TYR, tyrosinase; DCT, dopachrome tautomerase; <t>MITF,</t> melanocyte inducing transcription factor; TYRP1, tyrosinase related protein 1; PMEL, premelanosome protein. (I) Immunoblot analysis of melanogenesis-related proteins in primary human melanocytes treated as described in G. GAPDH served as a control. (J) Tyrosinase activity of A375 MC1R-KO cells rescued with different forms of MC1R ( n = 5 independent experiments). Cilio-MC1R was constructed by fusing the ciliary protein Arl13b with WT, R151C, or R160W MC1R. Cells were treated with or without 100 nM α-MSH for 36 h in the absence of serum and UV. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; CH, chloral hydrate; MC1R, melanocortin 1 receptor; UV, ultraviolet.
Rabbit Polyclonal Mitf Antibody, supplied by Cusabio, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+mitf/Rabbit+anti-+MITF+Polyclonal+Antibody/10__7324_slash_JAPS__2023__130107-74-1-17
Average 90 stars, based on 1 article reviews
rabbit polyclonal mitf antibody - by Bioz Stars, 2026-10
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nb100 56561af488  (Novus Biologicals)
88
Novus Biologicals nb100 56561af488
(A) Immunofluorescence images of PIG1 cells transfected <t>with</t> <t>MC1R-mEmerald</t> (green) and mock-treated, or treated with UV, 100 nM α-MSH, or both. Cells were treated with α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). Scale bar, 5 μm. (B) MC1R ciliary enrichment analysis of panel A as previously reported . Ciliary and cell body intensity of MC1R was measured using ImageJ. Enrichment at the cilium is expressed as: (integrated density at the cilium)/(integrated density in the cell body). Enrichment > 1 indicates higher localization of mEmerald tagged MC1R at the primary cilium than at the cell body. Every replicate was represented as a superplot. ( n = 30 ciliated cells from 3 different replicates) (C, D) Immunofluorescence images (C) of A375 MC1R-KO cells transfected with WT, R151C, or R160W MC1R-mEmerald (green) and mock-treated or treated with 100 nM α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). The percentage of ciliated cells with ciliary MC1R localization was quantified in panel D ( n = 3 independent experiments). Scale bar, 5 μm. (E) Immunofluorescence images of MC1R-mEmerald transfected primary human melanocytes treated with UV/α-MSH (100 nM). Cells were treated with α-MSH for 36 h in the presence of serum after UV exposure. Cells were stained with the Arl13b (red) and TYRP1 (magenta) antibodies. Nuclei were stained with DAPI (blue). Representative images for non-ciliated cells, ciliated cells without ciliary MC1R (MC1R − cilium), and ciliated cells with ciliary MC1R (MC1R + cilium) were selected from the same slides. Scale bar, 10 μm. (F) Quantification of the number of TYRP1 puncta ( n = 20 cells) as described in E. (G) Tyrosinase activity of primary human melanocytes that were mock treated, treated with 100 nM α-MSH, 100 nM α-MSH/30 μm ciliobrevin A (Cilio A), or 100 nM α-MSH/2 mM CH for 36 h in the absence of serum after UV exposure ( n = 3 independent experiments). (H) Quantitative RT-PCR analysis of melanogenesis-related genes in primary human melanocytes treated as described in G ( n = 3 independent experiments). TYR, tyrosinase; DCT, dopachrome tautomerase; <t>MITF,</t> melanocyte inducing transcription factor; TYRP1, tyrosinase related protein 1; PMEL, premelanosome protein. (I) Immunoblot analysis of melanogenesis-related proteins in primary human melanocytes treated as described in G. GAPDH served as a control. (J) Tyrosinase activity of A375 MC1R-KO cells rescued with different forms of MC1R ( n = 5 independent experiments). Cilio-MC1R was constructed by fusing the ciliary protein Arl13b with WT, R151C, or R160W MC1R. Cells were treated with or without 100 nM α-MSH for 36 h in the absence of serum and UV. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; CH, chloral hydrate; MC1R, melanocortin 1 receptor; UV, ultraviolet.
Nb100 56561af488, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+mitf/MITF+Antibody+(21D1418)+%5BAlexa+Fluor%C2%AE+488%5D/pmc06075866-249-9-6
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mitf  (St Johns Laboratory)
90
St Johns Laboratory mitf
(A) Immunofluorescence images of PIG1 cells transfected <t>with</t> <t>MC1R-mEmerald</t> (green) and mock-treated, or treated with UV, 100 nM α-MSH, or both. Cells were treated with α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). Scale bar, 5 μm. (B) MC1R ciliary enrichment analysis of panel A as previously reported . Ciliary and cell body intensity of MC1R was measured using ImageJ. Enrichment at the cilium is expressed as: (integrated density at the cilium)/(integrated density in the cell body). Enrichment > 1 indicates higher localization of mEmerald tagged MC1R at the primary cilium than at the cell body. Every replicate was represented as a superplot. ( n = 30 ciliated cells from 3 different replicates) (C, D) Immunofluorescence images (C) of A375 MC1R-KO cells transfected with WT, R151C, or R160W MC1R-mEmerald (green) and mock-treated or treated with 100 nM α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). The percentage of ciliated cells with ciliary MC1R localization was quantified in panel D ( n = 3 independent experiments). Scale bar, 5 μm. (E) Immunofluorescence images of MC1R-mEmerald transfected primary human melanocytes treated with UV/α-MSH (100 nM). Cells were treated with α-MSH for 36 h in the presence of serum after UV exposure. Cells were stained with the Arl13b (red) and TYRP1 (magenta) antibodies. Nuclei were stained with DAPI (blue). Representative images for non-ciliated cells, ciliated cells without ciliary MC1R (MC1R − cilium), and ciliated cells with ciliary MC1R (MC1R + cilium) were selected from the same slides. Scale bar, 10 μm. (F) Quantification of the number of TYRP1 puncta ( n = 20 cells) as described in E. (G) Tyrosinase activity of primary human melanocytes that were mock treated, treated with 100 nM α-MSH, 100 nM α-MSH/30 μm ciliobrevin A (Cilio A), or 100 nM α-MSH/2 mM CH for 36 h in the absence of serum after UV exposure ( n = 3 independent experiments). (H) Quantitative RT-PCR analysis of melanogenesis-related genes in primary human melanocytes treated as described in G ( n = 3 independent experiments). TYR, tyrosinase; DCT, dopachrome tautomerase; <t>MITF,</t> melanocyte inducing transcription factor; TYRP1, tyrosinase related protein 1; PMEL, premelanosome protein. (I) Immunoblot analysis of melanogenesis-related proteins in primary human melanocytes treated as described in G. GAPDH served as a control. (J) Tyrosinase activity of A375 MC1R-KO cells rescued with different forms of MC1R ( n = 5 independent experiments). Cilio-MC1R was constructed by fusing the ciliary protein Arl13b with WT, R151C, or R160W MC1R. Cells were treated with or without 100 nM α-MSH for 36 h in the absence of serum and UV. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; CH, chloral hydrate; MC1R, melanocortin 1 receptor; UV, ultraviolet.
Mitf, supplied by St Johns Laboratory, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+mitf/Anti-MITF+Antibody/pm34603597-117-9-11
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goat anti mitf antibody  (R&D Systems)
93
R&D Systems goat anti mitf antibody
Figure 3. Expression of melanocyte markers in ChMC. Mouse ChMC were examined for expression of <t>Mitf,</t> Mc1r, Trp2, Mlana, Slc, P-Mel, Pan Cytokeratin (RPE marker), c-Kit, <t>and</t> <t>S100β</t> using flow cytometry. The shaded areas show staining in the absence of primary antibody (secondary control), and the unshaded peaks show staining with primary antibody. The difference between the geometric means of primary antibody-stained cells and secondary antibody-stained control cells can be found in the top right corner of each graph. These experiments were performed at least 2 times with 3 different isolations of ChMC with similar results.
Goat Anti Mitf Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+mitf/Human+MITF+Antibody/pm40358170-102-15-19
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anti mitf nb110 10872 antibody  (Novus Biologicals)
91
Novus Biologicals anti mitf nb110 10872 antibody
Figure 3. Expression of melanocyte markers in ChMC. Mouse ChMC were examined for expression of <t>Mitf,</t> Mc1r, Trp2, Mlana, Slc, P-Mel, Pan Cytokeratin (RPE marker), c-Kit, <t>and</t> <t>S100β</t> using flow cytometry. The shaded areas show staining in the absence of primary antibody (secondary control), and the unshaded peaks show staining with primary antibody. The difference between the geometric means of primary antibody-stained cells and secondary antibody-stained control cells can be found in the top right corner of each graph. These experiments were performed at least 2 times with 3 different isolations of ChMC with similar results.
Anti Mitf Nb110 10872 Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


FIGURE 1. FUS is in a complex with MITF. A, Coomassie-stained FLAG-immunoprecipitated samples run on an 8.5% SDS-polyacrylamide gel; lane 1 (mock), FLAG-immunoprecipitated lysates from parental RAW264.7 C4 cell line; lane 2 (FM), FLAG immunoprecipitated lysates from RAW264.7 C4 cell line stably expressing FLAG-MITF. Lane 3 (M) marker, FUS and MITF based on size are indicated on the right. B, pulldown of RAW264.7 C4 cell lysates using GSH-Sepharose beads incubated either with GST (lane 1) or GST-MITF (lane 2) and probed with anti-FUS antibody. Input controls for RAW264.7 C4 lysates and GST beads are depicted in middle and bottom panels, respectively. C, top panel, schematic diagram of full-length and N- and C-terminal FUS constructs used for MITF interaction mapping. Ser-, Tyr-, Gly-, and Gln-rich N-terminal activation domain (AD) and C-terminal RGG-rich RNA binding domains (RBD) are shown. Bottom panel, coimmunoprecipitation studies in COS-7 cell lysates using full-length FLAG-MITF (lanes 2–4), full-length V5-FUS (lane 1 and 2), V5-tagged N-terminal FUS (lane 3, N; aa 1–259) or V5-tagged C-terminal FUS (lane 4, C; aa 260–518) immunoprecipitated (IP) with anti-FLAG antibody and probed with anti-V5 antibody. Input controls for V5-FUS and FLAG-MITF are shown. D, top panel, schematic diagram of full-length and N- and C-terminal MITF constructs used for FUS interactionmapping.N-terminalactivationdomains(AD1andAD2denotedasAD),basic(B),helix-loop-helix(HLH),andleucinezipper(LZ)domainsareshown. Ser-73 ERK phosphorylation site and Ser-307 p38 MAPK phosphorylation site as well as Lys-182 and Lys-316 sumoylation sites are also denoted. Bottom panel, immunoprecipitates pulled with FLAG antibody from COS-7 cell lysates expressing full-length FLAG-MITF (lane 1 and 3), full-length V5-FUS (lanes 2–5), FLAG-tagged N-terminal (aa 1–178) and C-terminal (aa 178–419) MITF (lanes 4 and 5, respectively) and immunoblotted (IB) with anti-V5 antibody. Loading controls for V5-FUS and FLAG-MITF are shown at the bottom.

Journal: Journal of Biological Chemistry

Article Title: The Multifunctional Protein Fused in Sarcoma (FUS) Is a Coactivator of Microphthalmia-associated Transcription Factor (MITF)

doi: 10.1074/jbc.m113.493874

Figure Lengend Snippet: FIGURE 1. FUS is in a complex with MITF. A, Coomassie-stained FLAG-immunoprecipitated samples run on an 8.5% SDS-polyacrylamide gel; lane 1 (mock), FLAG-immunoprecipitated lysates from parental RAW264.7 C4 cell line; lane 2 (FM), FLAG immunoprecipitated lysates from RAW264.7 C4 cell line stably expressing FLAG-MITF. Lane 3 (M) marker, FUS and MITF based on size are indicated on the right. B, pulldown of RAW264.7 C4 cell lysates using GSH-Sepharose beads incubated either with GST (lane 1) or GST-MITF (lane 2) and probed with anti-FUS antibody. Input controls for RAW264.7 C4 lysates and GST beads are depicted in middle and bottom panels, respectively. C, top panel, schematic diagram of full-length and N- and C-terminal FUS constructs used for MITF interaction mapping. Ser-, Tyr-, Gly-, and Gln-rich N-terminal activation domain (AD) and C-terminal RGG-rich RNA binding domains (RBD) are shown. Bottom panel, coimmunoprecipitation studies in COS-7 cell lysates using full-length FLAG-MITF (lanes 2–4), full-length V5-FUS (lane 1 and 2), V5-tagged N-terminal FUS (lane 3, N; aa 1–259) or V5-tagged C-terminal FUS (lane 4, C; aa 260–518) immunoprecipitated (IP) with anti-FLAG antibody and probed with anti-V5 antibody. Input controls for V5-FUS and FLAG-MITF are shown. D, top panel, schematic diagram of full-length and N- and C-terminal MITF constructs used for FUS interactionmapping.N-terminalactivationdomains(AD1andAD2denotedasAD),basic(B),helix-loop-helix(HLH),andleucinezipper(LZ)domainsareshown. Ser-73 ERK phosphorylation site and Ser-307 p38 MAPK phosphorylation site as well as Lys-182 and Lys-316 sumoylation sites are also denoted. Bottom panel, immunoprecipitates pulled with FLAG antibody from COS-7 cell lysates expressing full-length FLAG-MITF (lane 1 and 3), full-length V5-FUS (lanes 2–5), FLAG-tagged N-terminal (aa 1–178) and C-terminal (aa 178–419) MITF (lanes 4 and 5, respectively) and immunoblotted (IB) with anti-V5 antibody. Loading controls for V5-FUS and FLAG-MITF are shown at the bottom.

Article Snippet: Antibodies used were as follows: FLAG (mouse monoclonal, M2; Santa Cruz Biotechnology); V5 (mouse monoclonal, Invitrogen); FUS (rabbit polyclonal, Bethyl Laboratories, Inc.); GST (mouse monoclonal, Sigma); MITF and MITF PS307 (rabbit polyclonal affinity purified (5)); MITF (mouse monoclonal, Abcam), c-MYC (mouse monoclonal, Santa Cruz Biotechnology); HA (mouse monoclonal, Sigma); BRG1 (rabbit polyclonal affinity purified (24)); SUMO1 (mouse monoclonal, Santa Cruz Biotechnology); pp38 (rabbit polyclonal, Cell Signaling), and GAPDH (rabbit polyclonal, Santa Cruz Biotechnology).

Techniques: Staining, Immunoprecipitation, Stable Transfection, Expressing, Marker, Incubation, Construct, Activation Assay, RNA Binding Assay, Phospho-proteomics

FIGURE 2. FUS-MITF complex formation is mediated by CSF-1/RANKL signaling. A, immunoprecipitation (IP) of endogenous MITF from murine bone marrow- derivedmyeloidcellstreatedwithCSF-1alone(lane1)orCSF-1andRANKLfor24and72h(lanes2and3,respectively)andimmunoblotted(IB)withFUSantibody(top panel).InputcontrolsforFUSandMITFforthesametreatmentsareshowninthemiddleandbottompanels,respectively.B,immunoprecipitationofendogenousFUS and BRG1 using anti-FLAG antibody in RAW264.7 C4 cells stably expressing FLAG-MITF in response to CSF-1 and RANKL treatments. Lane 1, cells treated with CSF-1 alone; lanes 2–5, cells treated with CSF-1 and RANKL for 6, 24, 48, and 72 h, respectively. Top two panels show the immunoblotting with FUS- and BRG1-specific antibodies on immunoprecipitates. Loading controls for FUS, BRG1, and FLAG-MITF are shown in the bottom three panels. C, RAW264.7 C4 cells were transiently transfectedwithFLAG-taggedwildtype(wt,leftpanel)orS307AformofMITF(rightpanel),andtheinteractionofFUSwasexaminedwithrespecttoCSF-1andRANKL treatmentovertheindicatedtimecourse(top)byimmunoprecipitationwithFLAGantibodyfollowedbyimmunoblottingwithFUSantibody.Lane1,transfectedcells as above treated with CSF-1 alone;lanes 2–4,transfected cells treated with CSF-1 and RANKL for 1, 8, and 16 h, respectively. Input controls for FUS and FLAG-MITF are depictedinlowertwopanels.D,lysatesfromCOS-7cellsoverexpressingFLAG-MITF(lanes2–4),V5-FUS(lanes1–4),andeitherconstitutivelyactiveMYC-RAF(lane3)or MKK6 (lane 4) were immunoprecipitated with anti-FLAG antibody (IP: FLAG) and immunoblotted with anti-V5 antibody (top panel). Whole cell lysates were also immunoblotted with anti-V5, anti-MYC, anti-pp38, or anti-FLAG antibodies (input controls, bottom four panels).

Journal: Journal of Biological Chemistry

Article Title: The Multifunctional Protein Fused in Sarcoma (FUS) Is a Coactivator of Microphthalmia-associated Transcription Factor (MITF)

doi: 10.1074/jbc.m113.493874

Figure Lengend Snippet: FIGURE 2. FUS-MITF complex formation is mediated by CSF-1/RANKL signaling. A, immunoprecipitation (IP) of endogenous MITF from murine bone marrow- derivedmyeloidcellstreatedwithCSF-1alone(lane1)orCSF-1andRANKLfor24and72h(lanes2and3,respectively)andimmunoblotted(IB)withFUSantibody(top panel).InputcontrolsforFUSandMITFforthesametreatmentsareshowninthemiddleandbottompanels,respectively.B,immunoprecipitationofendogenousFUS and BRG1 using anti-FLAG antibody in RAW264.7 C4 cells stably expressing FLAG-MITF in response to CSF-1 and RANKL treatments. Lane 1, cells treated with CSF-1 alone; lanes 2–5, cells treated with CSF-1 and RANKL for 6, 24, 48, and 72 h, respectively. Top two panels show the immunoblotting with FUS- and BRG1-specific antibodies on immunoprecipitates. Loading controls for FUS, BRG1, and FLAG-MITF are shown in the bottom three panels. C, RAW264.7 C4 cells were transiently transfectedwithFLAG-taggedwildtype(wt,leftpanel)orS307AformofMITF(rightpanel),andtheinteractionofFUSwasexaminedwithrespecttoCSF-1andRANKL treatmentovertheindicatedtimecourse(top)byimmunoprecipitationwithFLAGantibodyfollowedbyimmunoblottingwithFUSantibody.Lane1,transfectedcells as above treated with CSF-1 alone;lanes 2–4,transfected cells treated with CSF-1 and RANKL for 1, 8, and 16 h, respectively. Input controls for FUS and FLAG-MITF are depictedinlowertwopanels.D,lysatesfromCOS-7cellsoverexpressingFLAG-MITF(lanes2–4),V5-FUS(lanes1–4),andeitherconstitutivelyactiveMYC-RAF(lane3)or MKK6 (lane 4) were immunoprecipitated with anti-FLAG antibody (IP: FLAG) and immunoblotted with anti-V5 antibody (top panel). Whole cell lysates were also immunoblotted with anti-V5, anti-MYC, anti-pp38, or anti-FLAG antibodies (input controls, bottom four panels).

Article Snippet: Antibodies used were as follows: FLAG (mouse monoclonal, M2; Santa Cruz Biotechnology); V5 (mouse monoclonal, Invitrogen); FUS (rabbit polyclonal, Bethyl Laboratories, Inc.); GST (mouse monoclonal, Sigma); MITF and MITF PS307 (rabbit polyclonal affinity purified (5)); MITF (mouse monoclonal, Abcam), c-MYC (mouse monoclonal, Santa Cruz Biotechnology); HA (mouse monoclonal, Sigma); BRG1 (rabbit polyclonal affinity purified (24)); SUMO1 (mouse monoclonal, Santa Cruz Biotechnology); pp38 (rabbit polyclonal, Cell Signaling), and GAPDH (rabbit polyclonal, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Stable Transfection, Expressing, Western Blot, Transfection

FIGURE 3. FUS is a coactivator of MITF in CSF-1/RANKL-mediated induc- tion of MITF target genes. A, soluble chromatin from BMMs treated either with CSF-1 alone or CSF-1 and RANKL for the indicated time points were immunoprecipitated with FUS antibody, and enrichment of Acp5 and Ctsk promoters was analyzed by qPCR. The top panel shows the relative positions of the oligonucleotides used with respect to the TSS of each gene. B, evalua- tion of FUS knockdown by shRNA FUS constructs. Left panel, either scrambled or shFUS retroviral constructs were nucleofected into RAW264.7 C4 F-M cells, and24hpost-infection,celllysateswereprobedforFUSproteinandTUBULIN protein (loading control). Right panel, mRNA levels of Fus in presence and absence of CSF-1 and RANKL 24 h post-infection with either scrambled (Scr) or shRNA FUS by qRT-PCR. C, RAW264.7 C4 F-M cells infected and treated as in B were analyzed for mRNA expression of Acp5 and Ctsk in the presence or absenceofCSF-1andRANKLbyqRT-PCR.Allexperimentsweretheaverageof at least two independent biological sets done in duplicate, and the error bars indicate S.D.

Journal: Journal of Biological Chemistry

Article Title: The Multifunctional Protein Fused in Sarcoma (FUS) Is a Coactivator of Microphthalmia-associated Transcription Factor (MITF)

doi: 10.1074/jbc.m113.493874

Figure Lengend Snippet: FIGURE 3. FUS is a coactivator of MITF in CSF-1/RANKL-mediated induc- tion of MITF target genes. A, soluble chromatin from BMMs treated either with CSF-1 alone or CSF-1 and RANKL for the indicated time points were immunoprecipitated with FUS antibody, and enrichment of Acp5 and Ctsk promoters was analyzed by qPCR. The top panel shows the relative positions of the oligonucleotides used with respect to the TSS of each gene. B, evalua- tion of FUS knockdown by shRNA FUS constructs. Left panel, either scrambled or shFUS retroviral constructs were nucleofected into RAW264.7 C4 F-M cells, and24hpost-infection,celllysateswereprobedforFUSproteinandTUBULIN protein (loading control). Right panel, mRNA levels of Fus in presence and absence of CSF-1 and RANKL 24 h post-infection with either scrambled (Scr) or shRNA FUS by qRT-PCR. C, RAW264.7 C4 F-M cells infected and treated as in B were analyzed for mRNA expression of Acp5 and Ctsk in the presence or absenceofCSF-1andRANKLbyqRT-PCR.Allexperimentsweretheaverageof at least two independent biological sets done in duplicate, and the error bars indicate S.D.

Article Snippet: Antibodies used were as follows: FLAG (mouse monoclonal, M2; Santa Cruz Biotechnology); V5 (mouse monoclonal, Invitrogen); FUS (rabbit polyclonal, Bethyl Laboratories, Inc.); GST (mouse monoclonal, Sigma); MITF and MITF PS307 (rabbit polyclonal affinity purified (5)); MITF (mouse monoclonal, Abcam), c-MYC (mouse monoclonal, Santa Cruz Biotechnology); HA (mouse monoclonal, Sigma); BRG1 (rabbit polyclonal affinity purified (24)); SUMO1 (mouse monoclonal, Santa Cruz Biotechnology); pp38 (rabbit polyclonal, Cell Signaling), and GAPDH (rabbit polyclonal, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Knockdown, shRNA, Construct, Retroviral, Infection, Control, Quantitative RT-PCR, Expressing

FIGURE 4. FUS, MITF, and BRG1 complex formation is mediated by Ser(P)-307 of MITF. A, coimmunoprecipitation of V5-FUS in COS-7 cells overexpressing either BRG1 and FLAG-MITF (lane 1), BRG1 and V5-FUS (lane 2), or all three constructs (lane 3) and immunoblotted (IB) with anti-BRG1 antibody (top panel) or anti-FLAG antibody (2nd panel from top). Input lysates for V5, BRG1, and FLAG-MITF are denoted in subsequent panels. B, coimmunoprecipitation of FLAG- tagged wild type MITF (lanes 2 and 4) or S307A mutant MITF (lanes 3 and 5) in the absence (lanes 1–3) or presence (lanes 4 and 5) of BRG1. Top panel shows immunoblot of V5 on FLAG immunoprecipitates (IP), and subsequent panels depict input controls. C, relative quantification of the band areas in B (top panel) using ImageJ software. D, relative mRNA expression of Acp5, Ctsk, and Fus in RAW264.7 C4 cells that were nucleofected with either plasmid encoding V5-Fus alone, Brg1 alone, or both plasmids. Post-nucleofection the cells were incubated in the presence (dark bars) or absence (light bars) of CSF-1 and RANKL for 24 h. All experiments were performed on at least two biological sets done in duplicate, and the error bars indicate S.D. E, soluble chromatin from RAW264.7 C4 cells nucleofected with either scrambled (Scr) or shFUS retroviral constructs and treated with CSF-1 and RANKL for 3 days were immunoprecipitated with either FUS antibody (left panel) or BRG1 antibody (right panel), and enrichment of the Ctsk promoter was analyzed by qPCR. All experiments were performed on at least two biological sets done in duplicate, and the error bars indicate S.D.

Journal: Journal of Biological Chemistry

Article Title: The Multifunctional Protein Fused in Sarcoma (FUS) Is a Coactivator of Microphthalmia-associated Transcription Factor (MITF)

doi: 10.1074/jbc.m113.493874

Figure Lengend Snippet: FIGURE 4. FUS, MITF, and BRG1 complex formation is mediated by Ser(P)-307 of MITF. A, coimmunoprecipitation of V5-FUS in COS-7 cells overexpressing either BRG1 and FLAG-MITF (lane 1), BRG1 and V5-FUS (lane 2), or all three constructs (lane 3) and immunoblotted (IB) with anti-BRG1 antibody (top panel) or anti-FLAG antibody (2nd panel from top). Input lysates for V5, BRG1, and FLAG-MITF are denoted in subsequent panels. B, coimmunoprecipitation of FLAG- tagged wild type MITF (lanes 2 and 4) or S307A mutant MITF (lanes 3 and 5) in the absence (lanes 1–3) or presence (lanes 4 and 5) of BRG1. Top panel shows immunoblot of V5 on FLAG immunoprecipitates (IP), and subsequent panels depict input controls. C, relative quantification of the band areas in B (top panel) using ImageJ software. D, relative mRNA expression of Acp5, Ctsk, and Fus in RAW264.7 C4 cells that were nucleofected with either plasmid encoding V5-Fus alone, Brg1 alone, or both plasmids. Post-nucleofection the cells were incubated in the presence (dark bars) or absence (light bars) of CSF-1 and RANKL for 24 h. All experiments were performed on at least two biological sets done in duplicate, and the error bars indicate S.D. E, soluble chromatin from RAW264.7 C4 cells nucleofected with either scrambled (Scr) or shFUS retroviral constructs and treated with CSF-1 and RANKL for 3 days were immunoprecipitated with either FUS antibody (left panel) or BRG1 antibody (right panel), and enrichment of the Ctsk promoter was analyzed by qPCR. All experiments were performed on at least two biological sets done in duplicate, and the error bars indicate S.D.

Article Snippet: Antibodies used were as follows: FLAG (mouse monoclonal, M2; Santa Cruz Biotechnology); V5 (mouse monoclonal, Invitrogen); FUS (rabbit polyclonal, Bethyl Laboratories, Inc.); GST (mouse monoclonal, Sigma); MITF and MITF PS307 (rabbit polyclonal affinity purified (5)); MITF (mouse monoclonal, Abcam), c-MYC (mouse monoclonal, Santa Cruz Biotechnology); HA (mouse monoclonal, Sigma); BRG1 (rabbit polyclonal affinity purified (24)); SUMO1 (mouse monoclonal, Santa Cruz Biotechnology); pp38 (rabbit polyclonal, Cell Signaling), and GAPDH (rabbit polyclonal, Santa Cruz Biotechnology).

Techniques: Construct, Mutagenesis, Western Blot, Quantitative Proteomics, Software, Expressing, Plasmid Preparation, Incubation, Retroviral, Immunoprecipitation

FIGURE 5. MITF phospho-sumoylation switch is regulated by CSF-1 and RANKL signaling. A, cell extracts prepared under denaturing lysis conditions from primary BMMs treated with either CSF1 (lane 1) or CSF1/RANKL (lane 2) for 72 h or COS-7 cells overexpressing FLAG-MITF and HA-SUMO (lane 3) were immunoblotted with anti-MITF antibody (top panel). GAPDH loading control is shown in the bottom panel. B, GSH-Sepharose pulldowns from RAW264.7 C4 cell lysates overexpressing GST-MITF and HA-SUMO1 treated with CSF-1 and RANKL for times (in hours) indicated below were immunoblotted (IB) with SUMO1 antibody and GST antibody (1st and 2nd panels from left). Input MITF and HA are denoted on right two panels. C, GST pulldown using COS-7 cells overexpressing GST-MITFalone(lane1),GST-MITFandHA-SUMO1(lane2),orGST-MITFandHA-SUMO1withconstitutivelyactiveMYC-RAForMKK6(lanes3and4,respectively) were analyzed by immunoblotting with GST antibody (top panel) or SUMO1 antibody (2nd from top). Inputs probed for phosphorylated p38 MAPK and MYC are shown in lower two panels.

Journal: Journal of Biological Chemistry

Article Title: The Multifunctional Protein Fused in Sarcoma (FUS) Is a Coactivator of Microphthalmia-associated Transcription Factor (MITF)

doi: 10.1074/jbc.m113.493874

Figure Lengend Snippet: FIGURE 5. MITF phospho-sumoylation switch is regulated by CSF-1 and RANKL signaling. A, cell extracts prepared under denaturing lysis conditions from primary BMMs treated with either CSF1 (lane 1) or CSF1/RANKL (lane 2) for 72 h or COS-7 cells overexpressing FLAG-MITF and HA-SUMO (lane 3) were immunoblotted with anti-MITF antibody (top panel). GAPDH loading control is shown in the bottom panel. B, GSH-Sepharose pulldowns from RAW264.7 C4 cell lysates overexpressing GST-MITF and HA-SUMO1 treated with CSF-1 and RANKL for times (in hours) indicated below were immunoblotted (IB) with SUMO1 antibody and GST antibody (1st and 2nd panels from left). Input MITF and HA are denoted on right two panels. C, GST pulldown using COS-7 cells overexpressing GST-MITFalone(lane1),GST-MITFandHA-SUMO1(lane2),orGST-MITFandHA-SUMO1withconstitutivelyactiveMYC-RAForMKK6(lanes3and4,respectively) were analyzed by immunoblotting with GST antibody (top panel) or SUMO1 antibody (2nd from top). Inputs probed for phosphorylated p38 MAPK and MYC are shown in lower two panels.

Article Snippet: Antibodies used were as follows: FLAG (mouse monoclonal, M2; Santa Cruz Biotechnology); V5 (mouse monoclonal, Invitrogen); FUS (rabbit polyclonal, Bethyl Laboratories, Inc.); GST (mouse monoclonal, Sigma); MITF and MITF PS307 (rabbit polyclonal affinity purified (5)); MITF (mouse monoclonal, Abcam), c-MYC (mouse monoclonal, Santa Cruz Biotechnology); HA (mouse monoclonal, Sigma); BRG1 (rabbit polyclonal affinity purified (24)); SUMO1 (mouse monoclonal, Santa Cruz Biotechnology); pp38 (rabbit polyclonal, Cell Signaling), and GAPDH (rabbit polyclonal, Santa Cruz Biotechnology).

Techniques: Lysis, Control, Western Blot

FIGURE 6. Sumoylation of MITF at Lys-316 inhibits interaction of FUS with MITF. A, cell extracts from either FLAG-MITF (wt) (lanes 1, 2, and 6) or FLAG-MITF with point or double mutations (K182R (lane 3), K316R (lane 4), K182R/K316R (K182/316R; lane 5), HA-SUMO1 (lanes 2–6), and MKK6 (lane 6)) expressing COS-7 cells were immunoprecipitated (IP) with anti-FLAG antibody (IP: FLAG). Immunoprecipitates were resolved by SDS-PAGE and immunoblotted (IB) with anti-HA antibody. Whole cell lysates were also immunoblotted with anti-FLAG, anti-HA, or anti-Pp38 antibody (input controls). B, cell extracts from either FLAG-MITF (wt) (lanes 2 and 7), FLAG-MITF K182R (K182R; lanes 3 and 8), FLAG-MITF K316R (K316R; lanes 4 and 9), FLAG-MITF K182R/K316R (K182/316R; lanes 5 and 10), V5-FUS (lanes 1–10), and HA-SUMO1 (lanes 6–10)-expressing COS-7 cells were immunoprecipitated with anti-FLAG antibody (IP: FLAG). Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-V5 or anti-FLAG antibody. Whole cell lysates were also immunoblotted with anti-V5 antibody (lower panel). C, lysates from COS-7 cells expressing FLAG-MITF (wt) (lane 2 and 4) or K316R FLAG-MITF mutant (lane 3 and 5) and V5-FUS (lanes 1–5) and HA-SUMO1 (lane 4 and 5) were immunoprecipitated with anti-FLAG antibody (IP: FLAG) and immunoblotted anti-BRG1 antibody (top panel). Whole cell lysates were also immunoblotted with anti-FLAG, anti-V5, or anti-BRG1 antibodies (input controls, bottom three panels).

Journal: Journal of Biological Chemistry

Article Title: The Multifunctional Protein Fused in Sarcoma (FUS) Is a Coactivator of Microphthalmia-associated Transcription Factor (MITF)

doi: 10.1074/jbc.m113.493874

Figure Lengend Snippet: FIGURE 6. Sumoylation of MITF at Lys-316 inhibits interaction of FUS with MITF. A, cell extracts from either FLAG-MITF (wt) (lanes 1, 2, and 6) or FLAG-MITF with point or double mutations (K182R (lane 3), K316R (lane 4), K182R/K316R (K182/316R; lane 5), HA-SUMO1 (lanes 2–6), and MKK6 (lane 6)) expressing COS-7 cells were immunoprecipitated (IP) with anti-FLAG antibody (IP: FLAG). Immunoprecipitates were resolved by SDS-PAGE and immunoblotted (IB) with anti-HA antibody. Whole cell lysates were also immunoblotted with anti-FLAG, anti-HA, or anti-Pp38 antibody (input controls). B, cell extracts from either FLAG-MITF (wt) (lanes 2 and 7), FLAG-MITF K182R (K182R; lanes 3 and 8), FLAG-MITF K316R (K316R; lanes 4 and 9), FLAG-MITF K182R/K316R (K182/316R; lanes 5 and 10), V5-FUS (lanes 1–10), and HA-SUMO1 (lanes 6–10)-expressing COS-7 cells were immunoprecipitated with anti-FLAG antibody (IP: FLAG). Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-V5 or anti-FLAG antibody. Whole cell lysates were also immunoblotted with anti-V5 antibody (lower panel). C, lysates from COS-7 cells expressing FLAG-MITF (wt) (lane 2 and 4) or K316R FLAG-MITF mutant (lane 3 and 5) and V5-FUS (lanes 1–5) and HA-SUMO1 (lane 4 and 5) were immunoprecipitated with anti-FLAG antibody (IP: FLAG) and immunoblotted anti-BRG1 antibody (top panel). Whole cell lysates were also immunoblotted with anti-FLAG, anti-V5, or anti-BRG1 antibodies (input controls, bottom three panels).

Article Snippet: Antibodies used were as follows: FLAG (mouse monoclonal, M2; Santa Cruz Biotechnology); V5 (mouse monoclonal, Invitrogen); FUS (rabbit polyclonal, Bethyl Laboratories, Inc.); GST (mouse monoclonal, Sigma); MITF and MITF PS307 (rabbit polyclonal affinity purified (5)); MITF (mouse monoclonal, Abcam), c-MYC (mouse monoclonal, Santa Cruz Biotechnology); HA (mouse monoclonal, Sigma); BRG1 (rabbit polyclonal affinity purified (24)); SUMO1 (mouse monoclonal, Santa Cruz Biotechnology); pp38 (rabbit polyclonal, Cell Signaling), and GAPDH (rabbit polyclonal, Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, SDS Page, Mutagenesis

Fig. 6 Enforced KDM5B expression facilitates melanocytic lineage-directed elimination by TMECG. a Quantitation of mRNA after 24 h, 48 h, 72 h and 7 days of Cpd1 treatment of MaMel63a cells as assessed by qPCR. Mean ± SD. Shown is one representative example. b Regulation of differentiation, cytokinesis, and mitotic spindle assembly genes as detected by cDNA microarray analysis after KDM5B shRNA knockdown in WM3734 cells (n = 1). c, d Immunoblotting of melanocytic lineage and (de-)differentiation markers after 24 h of KDM5B induction in WM3734Tet3G-KDM5B cells (c) and after 72 h of Cpd1 treatment in MaMel63a cells (d). Shown are representative data (n = 2). e Anti-MITF immunostaining (upper panel) and Fontana-Masson staining (lower panels) of CM melanoma tumor grafts from Cpd1-treated vs. control mice. f MTT cell viability assay of WM3734 cells. Representative example is shown left (mean ± SD, n = 2) and corresponding IC50 values on the right. TMECG was either concurrently given together with Cpd1 (“con”) or added 3 days after Cpd1 pre-treatment (“pre”). Readout was performed after 72 h of TMECG treatment. g Persister-state-directed therapy model in vivo. Left: schematic representation of treatment dosing and timing in immunodeficient NMRI-(nu/nu)-nude mice. Right: tumor volumes of WM3734 xenografts (endpoint at day 30). TMECG was either concurrently given together with Cpd1 (“con”) or added one week after Cpd1 pre-treatment (“pre”). Mean ±SEM (6 mice in TMECG and Cpd1 control group, five mice in “con” and seven mice in “pre” group). Significance was determined by two-sided Mann–Whitney test. Source data are provided as a Source Data file.

Journal: Nature communications

Article Title: Persister state-directed transitioning and vulnerability in melanoma.

doi: 10.1038/s41467-022-30641-9

Figure Lengend Snippet: Fig. 6 Enforced KDM5B expression facilitates melanocytic lineage-directed elimination by TMECG. a Quantitation of mRNA after 24 h, 48 h, 72 h and 7 days of Cpd1 treatment of MaMel63a cells as assessed by qPCR. Mean ± SD. Shown is one representative example. b Regulation of differentiation, cytokinesis, and mitotic spindle assembly genes as detected by cDNA microarray analysis after KDM5B shRNA knockdown in WM3734 cells (n = 1). c, d Immunoblotting of melanocytic lineage and (de-)differentiation markers after 24 h of KDM5B induction in WM3734Tet3G-KDM5B cells (c) and after 72 h of Cpd1 treatment in MaMel63a cells (d). Shown are representative data (n = 2). e Anti-MITF immunostaining (upper panel) and Fontana-Masson staining (lower panels) of CM melanoma tumor grafts from Cpd1-treated vs. control mice. f MTT cell viability assay of WM3734 cells. Representative example is shown left (mean ± SD, n = 2) and corresponding IC50 values on the right. TMECG was either concurrently given together with Cpd1 (“con”) or added 3 days after Cpd1 pre-treatment (“pre”). Readout was performed after 72 h of TMECG treatment. g Persister-state-directed therapy model in vivo. Left: schematic representation of treatment dosing and timing in immunodeficient NMRI-(nu/nu)-nude mice. Right: tumor volumes of WM3734 xenografts (endpoint at day 30). TMECG was either concurrently given together with Cpd1 (“con”) or added one week after Cpd1 pre-treatment (“pre”). Mean ±SEM (6 mice in TMECG and Cpd1 control group, five mice in “con” and seven mice in “pre” group). Significance was determined by two-sided Mann–Whitney test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies (Axl (8661), CDH2 (13116), tubulin (2148), ZEB1/TCF8 (3396), Smad1 (9743), and TJP1 (8193; all diluted 1:1000, all Cell Signaling, Cambridge, UK), FKBP12 (ab24373), histone H3 (ab1791, diluted 1:5000), histone H3K4me3 (ab8580, diluted 1:2000), MITF (ab80651, diluted 1:500), and Notch1 (ab52627, all diluted 1:1000, all Abcam, Cambridge, UK), KDM5B (NB100-97821, diluted 1:2000, Novus Biologicals, St. Louis, MO, USA) and GAPDH (SC-510, diluted 1:5000, Santa Cruz, Dallas, TX, USA)) were incubated overnight at 4 °C either in PBS containing 0.1% Tween-20 and 5% milk or in 1× Net-G buffer (10× Net-G contains 1.5 M NaCl, 50 mM EDTA, 500 mM Tris 0.5% Tween-20, and 0.4% gelatine).

Techniques: Expressing, Quantitation Assay, Microarray, shRNA, Knockdown, Western Blot, Immunostaining, Staining, Control, Viability Assay, In Vivo, MANN-WHITNEY

(A) Immunofluorescence images of PIG1 cells transfected with MC1R-mEmerald (green) and mock-treated, or treated with UV, 100 nM α-MSH, or both. Cells were treated with α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). Scale bar, 5 μm. (B) MC1R ciliary enrichment analysis of panel A as previously reported . Ciliary and cell body intensity of MC1R was measured using ImageJ. Enrichment at the cilium is expressed as: (integrated density at the cilium)/(integrated density in the cell body). Enrichment > 1 indicates higher localization of mEmerald tagged MC1R at the primary cilium than at the cell body. Every replicate was represented as a superplot. ( n = 30 ciliated cells from 3 different replicates) (C, D) Immunofluorescence images (C) of A375 MC1R-KO cells transfected with WT, R151C, or R160W MC1R-mEmerald (green) and mock-treated or treated with 100 nM α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). The percentage of ciliated cells with ciliary MC1R localization was quantified in panel D ( n = 3 independent experiments). Scale bar, 5 μm. (E) Immunofluorescence images of MC1R-mEmerald transfected primary human melanocytes treated with UV/α-MSH (100 nM). Cells were treated with α-MSH for 36 h in the presence of serum after UV exposure. Cells were stained with the Arl13b (red) and TYRP1 (magenta) antibodies. Nuclei were stained with DAPI (blue). Representative images for non-ciliated cells, ciliated cells without ciliary MC1R (MC1R − cilium), and ciliated cells with ciliary MC1R (MC1R + cilium) were selected from the same slides. Scale bar, 10 μm. (F) Quantification of the number of TYRP1 puncta ( n = 20 cells) as described in E. (G) Tyrosinase activity of primary human melanocytes that were mock treated, treated with 100 nM α-MSH, 100 nM α-MSH/30 μm ciliobrevin A (Cilio A), or 100 nM α-MSH/2 mM CH for 36 h in the absence of serum after UV exposure ( n = 3 independent experiments). (H) Quantitative RT-PCR analysis of melanogenesis-related genes in primary human melanocytes treated as described in G ( n = 3 independent experiments). TYR, tyrosinase; DCT, dopachrome tautomerase; MITF, melanocyte inducing transcription factor; TYRP1, tyrosinase related protein 1; PMEL, premelanosome protein. (I) Immunoblot analysis of melanogenesis-related proteins in primary human melanocytes treated as described in G. GAPDH served as a control. (J) Tyrosinase activity of A375 MC1R-KO cells rescued with different forms of MC1R ( n = 5 independent experiments). Cilio-MC1R was constructed by fusing the ciliary protein Arl13b with WT, R151C, or R160W MC1R. Cells were treated with or without 100 nM α-MSH for 36 h in the absence of serum and UV. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; CH, chloral hydrate; MC1R, melanocortin 1 receptor; UV, ultraviolet.

Journal: PLOS Biology

Article Title: Melanocortin 1 receptor mediates melanin production by interacting with the BBSome in primary cilia

doi: 10.1371/journal.pbio.3002940

Figure Lengend Snippet: (A) Immunofluorescence images of PIG1 cells transfected with MC1R-mEmerald (green) and mock-treated, or treated with UV, 100 nM α-MSH, or both. Cells were treated with α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). Scale bar, 5 μm. (B) MC1R ciliary enrichment analysis of panel A as previously reported . Ciliary and cell body intensity of MC1R was measured using ImageJ. Enrichment at the cilium is expressed as: (integrated density at the cilium)/(integrated density in the cell body). Enrichment > 1 indicates higher localization of mEmerald tagged MC1R at the primary cilium than at the cell body. Every replicate was represented as a superplot. ( n = 30 ciliated cells from 3 different replicates) (C, D) Immunofluorescence images (C) of A375 MC1R-KO cells transfected with WT, R151C, or R160W MC1R-mEmerald (green) and mock-treated or treated with 100 nM α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). The percentage of ciliated cells with ciliary MC1R localization was quantified in panel D ( n = 3 independent experiments). Scale bar, 5 μm. (E) Immunofluorescence images of MC1R-mEmerald transfected primary human melanocytes treated with UV/α-MSH (100 nM). Cells were treated with α-MSH for 36 h in the presence of serum after UV exposure. Cells were stained with the Arl13b (red) and TYRP1 (magenta) antibodies. Nuclei were stained with DAPI (blue). Representative images for non-ciliated cells, ciliated cells without ciliary MC1R (MC1R − cilium), and ciliated cells with ciliary MC1R (MC1R + cilium) were selected from the same slides. Scale bar, 10 μm. (F) Quantification of the number of TYRP1 puncta ( n = 20 cells) as described in E. (G) Tyrosinase activity of primary human melanocytes that were mock treated, treated with 100 nM α-MSH, 100 nM α-MSH/30 μm ciliobrevin A (Cilio A), or 100 nM α-MSH/2 mM CH for 36 h in the absence of serum after UV exposure ( n = 3 independent experiments). (H) Quantitative RT-PCR analysis of melanogenesis-related genes in primary human melanocytes treated as described in G ( n = 3 independent experiments). TYR, tyrosinase; DCT, dopachrome tautomerase; MITF, melanocyte inducing transcription factor; TYRP1, tyrosinase related protein 1; PMEL, premelanosome protein. (I) Immunoblot analysis of melanogenesis-related proteins in primary human melanocytes treated as described in G. GAPDH served as a control. (J) Tyrosinase activity of A375 MC1R-KO cells rescued with different forms of MC1R ( n = 5 independent experiments). Cilio-MC1R was constructed by fusing the ciliary protein Arl13b with WT, R151C, or R160W MC1R. Cells were treated with or without 100 nM α-MSH for 36 h in the absence of serum and UV. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; CH, chloral hydrate; MC1R, melanocortin 1 receptor; UV, ultraviolet.

Article Snippet: Primary antibodies used in this study included the following: β-actin (Abways, AB0035; 1:1,000), ace-tubulin (Sigma-Aldrich, T7451; 1:1,000), ATP1A1 (Proteintech, 14418-1-AP; 1:1,000), Arl13b (Proteintech, 17711-1-AP; 1:2,000), BBS1 (Proteintech, 21118-1-AP; 1:1,000), BBS2 (Proteintech, 11188-2-AP; 1:1,000), BBS4 (Proteintech, 12766-1-AP; 1:1,000), BBS9 (Proteintech, 14460-1-AP; 1:1,000), Flag (Sigma-Aldrich, F1804; 1:2,000; Proteintech, 20543-1-AP; 1:1,000), DCT (Santa Cruz Biotechnology, sc-74439; 1:1,000), GFP (Abways, AB0045; 1:1,000; Abways, AB0005; 1:1,000), HA (Sigma-Aldrich, H9658; 1:5,000), MC1R (Signalway Antibody, 36969; 1:500), MITF (Proteintech, 13092-1-AP; 1:1,000; Santa Cruz Biotechnology, sc-515925; 1:1,000), TYR (Santa Cruz Biotechnology, sc-20035; 1:1,000), TYRP1 (Santa Cruz Biotechnology, sc-166857; 1:1,000).

Techniques: Immunofluorescence, Transfection, Staining, Activity Assay, Quantitative RT-PCR, Western Blot, Control, Construct

(A, B) Detection of ciliary cAMP levels by cilia-targeted cADDis (green) and the reference ciliary marker 5HT6-mCherry (red) in A375 MC1R-KO cells rescued with MC1R or cilio-MC1R (constructed by fusing the ciliary protein Arl13b with MC1R). Cells were pre-serum-starved for 24 h and cultured in the absence UV. Panel A shows time-lapse images of cADDis (green) and cilia (red) after the addition of 1 μm α-MSH (denoted by arrow). Then, 100 μm forskolin was added at 6 min (denoted by arrow). Scale bar, 2.5 μm. In panel B, the fluorescence intensities were normalized to the time point of −1 min ( n = 5 ciliated cells from 5 independent experiments). The normalized fluorescence intensity is expressed as: (the fluorescence intensity of mCherry / the fluorescence intensity of cADDis) / (the fluorescence intensity of mCherry at −1 min / the fluorescence intensity of cADDis at −1 min). (C) Time course of total cellular cAMP levels of A375 MC1R-KO cells rescued with MC1R or cilio-MC1R upon stimulation with 1 μm α-MSH. Cells were pre-serum-starved for 24 h and cultured in the absence UV. (D) Volcano plots of differentially expressed genes in A375 MC1R-KO cells rescued with WT MC1R or cilio-MC1R. Cells were cultured in the absence of serum and UV, and treated with 100 nM α-MSH for 12 h before mRNA extraction. Genes with fold change >2 and adjusted p -value <0.05 were considered as differentially expressed. Melanogenesis-related genes were highlighted and marked on the spot. (E) Quantitative RT-PCR analysis of the indicated genes of A375 MC1R-KO cells transfected with the control vector, MC1R, or cilio-MC1R. Cell were treated with 100 nM α-MSH for 12 h before mRNA extraction ( n = 3 independent experiments). Cells were cultured in the absence of serum and UV. (F) Immunoblot analysis of the indicated proteins in A375 MC1R-KO cells rescued with the control vector, MC1R, or cilio-MC1R. Cells were treated with 100 nM α-MSH for 24 h in the absence of serum and UV before lysis. GAPDH served as a control. (G, H) Immunoprecipitation and immunoblotting showing the interaction between Sox-3×Flag and MITF-HA. HEK293T cells were transfected with the indicated plasmids and immunoprecipitated with antibodies against Flag (G) and HA (H). (I) Dual-luciferase reporter assay of the pGL3-basic or DCT promoter transfected with the indicated plasmids. The relative luciferase activity was calculated as the luciferase activity normalized with the control vector ( n = 3 independent experiments). (J) Tyrosinase activity of A375 MC1R-KO cells rescued with the control vector, MC1R, or cilio-MC1R. Cells were transfected with control or Sox9 shRNAs and treated with 100 nM α-MSH for 24 h in the absence of serum and UV before detection ( n = 3 independent experiments). (K) Immunoblot analysis of the indicated proteins in A375 MC1R-KO cells rescued with the control vector, MC1R, or cilio-MC1R. Cells were transfected with control or Sox9 shRNAs and treated with α-MSH for 24 h in the absence of serum and UV before lysis. GAPDH served as a control. Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t test (B and C) or one-way ANOVA (E, I, and J); * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; MC1R, melanocortin 1 receptor; UV, ultraviolet.

Journal: PLOS Biology

Article Title: Melanocortin 1 receptor mediates melanin production by interacting with the BBSome in primary cilia

doi: 10.1371/journal.pbio.3002940

Figure Lengend Snippet: (A, B) Detection of ciliary cAMP levels by cilia-targeted cADDis (green) and the reference ciliary marker 5HT6-mCherry (red) in A375 MC1R-KO cells rescued with MC1R or cilio-MC1R (constructed by fusing the ciliary protein Arl13b with MC1R). Cells were pre-serum-starved for 24 h and cultured in the absence UV. Panel A shows time-lapse images of cADDis (green) and cilia (red) after the addition of 1 μm α-MSH (denoted by arrow). Then, 100 μm forskolin was added at 6 min (denoted by arrow). Scale bar, 2.5 μm. In panel B, the fluorescence intensities were normalized to the time point of −1 min ( n = 5 ciliated cells from 5 independent experiments). The normalized fluorescence intensity is expressed as: (the fluorescence intensity of mCherry / the fluorescence intensity of cADDis) / (the fluorescence intensity of mCherry at −1 min / the fluorescence intensity of cADDis at −1 min). (C) Time course of total cellular cAMP levels of A375 MC1R-KO cells rescued with MC1R or cilio-MC1R upon stimulation with 1 μm α-MSH. Cells were pre-serum-starved for 24 h and cultured in the absence UV. (D) Volcano plots of differentially expressed genes in A375 MC1R-KO cells rescued with WT MC1R or cilio-MC1R. Cells were cultured in the absence of serum and UV, and treated with 100 nM α-MSH for 12 h before mRNA extraction. Genes with fold change >2 and adjusted p -value <0.05 were considered as differentially expressed. Melanogenesis-related genes were highlighted and marked on the spot. (E) Quantitative RT-PCR analysis of the indicated genes of A375 MC1R-KO cells transfected with the control vector, MC1R, or cilio-MC1R. Cell were treated with 100 nM α-MSH for 12 h before mRNA extraction ( n = 3 independent experiments). Cells were cultured in the absence of serum and UV. (F) Immunoblot analysis of the indicated proteins in A375 MC1R-KO cells rescued with the control vector, MC1R, or cilio-MC1R. Cells were treated with 100 nM α-MSH for 24 h in the absence of serum and UV before lysis. GAPDH served as a control. (G, H) Immunoprecipitation and immunoblotting showing the interaction between Sox-3×Flag and MITF-HA. HEK293T cells were transfected with the indicated plasmids and immunoprecipitated with antibodies against Flag (G) and HA (H). (I) Dual-luciferase reporter assay of the pGL3-basic or DCT promoter transfected with the indicated plasmids. The relative luciferase activity was calculated as the luciferase activity normalized with the control vector ( n = 3 independent experiments). (J) Tyrosinase activity of A375 MC1R-KO cells rescued with the control vector, MC1R, or cilio-MC1R. Cells were transfected with control or Sox9 shRNAs and treated with 100 nM α-MSH for 24 h in the absence of serum and UV before detection ( n = 3 independent experiments). (K) Immunoblot analysis of the indicated proteins in A375 MC1R-KO cells rescued with the control vector, MC1R, or cilio-MC1R. Cells were transfected with control or Sox9 shRNAs and treated with α-MSH for 24 h in the absence of serum and UV before lysis. GAPDH served as a control. Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t test (B and C) or one-way ANOVA (E, I, and J); * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; MC1R, melanocortin 1 receptor; UV, ultraviolet.

Article Snippet: Primary antibodies used in this study included the following: β-actin (Abways, AB0035; 1:1,000), ace-tubulin (Sigma-Aldrich, T7451; 1:1,000), ATP1A1 (Proteintech, 14418-1-AP; 1:1,000), Arl13b (Proteintech, 17711-1-AP; 1:2,000), BBS1 (Proteintech, 21118-1-AP; 1:1,000), BBS2 (Proteintech, 11188-2-AP; 1:1,000), BBS4 (Proteintech, 12766-1-AP; 1:1,000), BBS9 (Proteintech, 14460-1-AP; 1:1,000), Flag (Sigma-Aldrich, F1804; 1:2,000; Proteintech, 20543-1-AP; 1:1,000), DCT (Santa Cruz Biotechnology, sc-74439; 1:1,000), GFP (Abways, AB0045; 1:1,000; Abways, AB0005; 1:1,000), HA (Sigma-Aldrich, H9658; 1:5,000), MC1R (Signalway Antibody, 36969; 1:500), MITF (Proteintech, 13092-1-AP; 1:1,000; Santa Cruz Biotechnology, sc-515925; 1:1,000), TYR (Santa Cruz Biotechnology, sc-20035; 1:1,000), TYRP1 (Santa Cruz Biotechnology, sc-166857; 1:1,000).

Techniques: Marker, Construct, Cell Culture, Fluorescence, Extraction, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Western Blot, Lysis, Immunoprecipitation, Luciferase, Reporter Assay, Activity Assay, Two Tailed Test

Figure 3. Expression of melanocyte markers in ChMC. Mouse ChMC were examined for expression of Mitf, Mc1r, Trp2, Mlana, Slc, P-Mel, Pan Cytokeratin (RPE marker), c-Kit, and S100β using flow cytometry. The shaded areas show staining in the absence of primary antibody (secondary control), and the unshaded peaks show staining with primary antibody. The difference between the geometric means of primary antibody-stained cells and secondary antibody-stained control cells can be found in the top right corner of each graph. These experiments were performed at least 2 times with 3 different isolations of ChMC with similar results.

Journal: Cells

Article Title: Isolation and Characterization of Mouse Choroidal Melanocytes and Their Proinflammatory Characteristics.

doi: 10.3390/cells14090646

Figure Lengend Snippet: Figure 3. Expression of melanocyte markers in ChMC. Mouse ChMC were examined for expression of Mitf, Mc1r, Trp2, Mlana, Slc, P-Mel, Pan Cytokeratin (RPE marker), c-Kit, and S100β using flow cytometry. The shaded areas show staining in the absence of primary antibody (secondary control), and the unshaded peaks show staining with primary antibody. The difference between the geometric means of primary antibody-stained cells and secondary antibody-stained control cells can be found in the top right corner of each graph. These experiments were performed at least 2 times with 3 different isolations of ChMC with similar results.

Article Snippet: Choroidoscleral complex was then incubated with rabbit anti-S100β antibody (ab52642, Abcam, Waltham, MA, USA) and goat anti-Mitf antibody (AF5769-SP; R&D Systems) diluted 1/250 in the blocking buffer, and fresh antibodies were added daily for 3 days at room temperature on a shaker.

Techniques: Expressing, Marker, Flow Cytometry, Staining, Control

Figure 8. Immunostaining of the melanocytes in the choroidoscleral complex. Choroid/RPE whole- mounts were prepared from 8-week-old FVB/NJ mice and stained with specific antibodies as detailed in the Section 2. Melanocytes positive for S100β ((B); red) and MITF ((C), cyan or white) were detected in the choroidoscleral complex. A low magnification of wholemount double staining is shown in (A). Higher magnification of double staining is shown in (D). Images were captured using Nikon A1 confocal microscope and processed using NIS-Elements Software. Scale bar = 50 µm. These experiments were repeated with eyes from at least 5 mice with similar results.

Journal: Cells

Article Title: Isolation and Characterization of Mouse Choroidal Melanocytes and Their Proinflammatory Characteristics.

doi: 10.3390/cells14090646

Figure Lengend Snippet: Figure 8. Immunostaining of the melanocytes in the choroidoscleral complex. Choroid/RPE whole- mounts were prepared from 8-week-old FVB/NJ mice and stained with specific antibodies as detailed in the Section 2. Melanocytes positive for S100β ((B); red) and MITF ((C), cyan or white) were detected in the choroidoscleral complex. A low magnification of wholemount double staining is shown in (A). Higher magnification of double staining is shown in (D). Images were captured using Nikon A1 confocal microscope and processed using NIS-Elements Software. Scale bar = 50 µm. These experiments were repeated with eyes from at least 5 mice with similar results.

Article Snippet: Choroidoscleral complex was then incubated with rabbit anti-S100β antibody (ab52642, Abcam, Waltham, MA, USA) and goat anti-Mitf antibody (AF5769-SP; R&D Systems) diluted 1/250 in the blocking buffer, and fresh antibodies were added daily for 3 days at room temperature on a shaker.

Techniques: Immunostaining, Staining, Double Staining, Microscopy, Software