Hispolon was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Camptothecin, vitamin C, mushroom tyrosinase, L-DOPA, α-MSH, dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH), sodium dodecyl sulfate (SDS) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), α-modified essential medium (α-MEM), fetal bovine serum (FBS), penicillin, streptomycin and trypsin-EDTA were purchased from Gibco BRL/Invitrogen (Carlsbad, CA, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Affymetrix/USB (Cleveland, OH, USA). The β-arbutin was purchased from Alfa Aesar (Ward Hill, MA, USA). The anti-tyrosinase, anti-MITF and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The FITC-labeled annexin-V/PI apoptosis detection kit was purchased from BD Biosciences (San Jose, CA, USA). Caspases colorimetric assay kit was purchased from BioVision, Inc. (Milpitas, CA, USA). Deionized distilled water (ddH2O) for solutions and buffers was obtained from the Milli-Q system (Millipore, Bedford, MA, USA).
Anti mitf
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-MITF is a laboratory product that can be used for the detection of MITF (Microphthalmia-associated transcription factor) in a variety of research applications. MITF is a key regulator of melanocyte development and function, and is widely used as a marker in melanoma research and other related fields. The Anti-MITF product provides a specific and reliable tool for researchers to study MITF expression and its role in biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti tyrosinase, by Santa Cruz Biotechnology (6 mentions)
Mushroom tyrosinase, by Merck Group (4 mentions)
Anti trp 2, by Santa Cruz Biotechnology (4 mentions)
L dopa, by Merck Group (3 mentions)
α msh, by Merck Group (3 mentions)
Anti trp1, by Santa Cruz Biotechnology (3 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Milli q system, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Vitamin c, by Merck Group (1 mentions)
Camptothecin, by Merck Group (1 mentions)
Fitc labeled annexin 5 pi apoptosis detection kit, by BD (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (1 mentions)
Hispolon, by Enzo Life Sciences (1 mentions)
L 3 4 dihydroxyphenylalanine l dopa, by Merck Group (1 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
11 protocols using anti mitf
1
Hispolon Modulation of Melanogenesis and Cell Viability
2
Melanogenesis Regulation via Signaling Pathways
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Synthetic melanin, mushroom tyrosinase, L-3,4-Dihydroxyphenylalanine (L-DOPA), alpha-melanocyte stimulating hormone (α-MSH), arbutin, and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Anti-tyrosinase, anti-TRP-1, anti-TRP-2, anti-MITF, anti-c-AMP-dependent protein kinase (PKA), and anti-phospho-PKA (Thr198) antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-c-AMP-related element binding protein (CREB), anti-phospho-CREB (Ser133), anti-p38, anti-phospho-p38 (Thr180/Tyr182), anti-extracellular signal-related kinase 1/2 (ERK), anti-phospho-ERK (Thr202/Tyr204), anti-c-Jun-N-terminal kinase (JNK), anti-phopsho-JNK (Thr183/Tyr185), and anti-tubulin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). All of the other chemicals and solvents used were analytical grade.
3
Murine Monoclonal Antibodies for Immunodetection
4
Western Blot Analysis of Melanogenic Proteins
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Once the cells were harvested, the cell pellets were lysed in radioimmunoprecipitation (RIPA) buffer containing 1% NP-40, 1% sodium deoxycholate, and protease inhibitor (PI) cocktail on ice for 30 min. This was followed by centrifugation at 13,000 rpm for 30 min at 4 °C. The resulting supernatants were subsequently collected. Proteins were separated using 8% to 15% SDS polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% skim milk for 1 h at room temperature, followed by incubation with a primary antibody. Anti-Tyrosinase, anti-TRP1, anti-TRP2, anti-MITF, anti-phospho AKT, anti-AKT, anti-phospho ERK, anti-ERK, anti-phospho CREB, anti-CREB, and anti-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated with donkey anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated IgG secondary antibody for 2 h. Protein bands were detected by an ImageQuant LAS 4000 mini (Fujifilm, Tokyo, Japan) and visualized using an image analysis program (Multi Gauge Ver. 3.0, Fujifilm, Tokyo, Japan).
5
Immunofluorescence Assay for Cell Markers
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Cells were fixed with 4% paraformaldehyde for 20 min at room temperature followed by 0.1% Triton X-100 to allow cell permeabilization. Cells were then incubated with the following primary antibodies: anti-PPARγ, anti-SREBP1, anti-RUNX2, anti-Mitf, anti-Tyrosinase, anti-TRP2 (Santa Cruz Biotechnology, USA), anti-Sox2, anti-Nestin antibody (Merck Millipore, Germany), for 1 hour. Primary antibodies were visualized using anti-rabbit IgG, anti-goat or anti-mouse IgG Alexa Fluor 488 (BD Bioscences). Nuclei were visualized with DAPI. Fluorescence signals were recorded using a CCD camera (Zeiss, Oberkochen, Germany).
6
Melanogenesis Regulation Molecular Markers
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Primary antibodies for mouse anti-MITF (#sc-25386), tyrosinase (#sc-15341), TRP-1 (#sc-514900), TRP-2 (#sc-74439), p-ERK (#sc-7383), ERK 1/2 (#sc-514302), and β-actin (#sc-47778) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary anti-bodies for mouse anti-cyclic AMP (cAMP) response element-binding protein (CREB) (8763) (#9198S) and CREB (D76D11) (#4820S) were purchased from Cell Signaling Technology (Beverly, MA, USA); all primary antibodies were used at 1:1000 dilutions. Secondary mouse antibody for primary antibodies was purchased from Enzo Life Sciences (Farmingdale, NY, USA) and used at 1:5000 dilutions. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and penicillin/streptomycin were purchased from Gibco BRL (Carlsbad, CA, USA). Most chemicals, including α-MSH, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), L-DOPA, 1,1-diphenyl-2-picryl-hydrazy (DPPH), 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), arbutin, and mushroom tyrosinase were purchased from Sigma-Aldrich (St. Louis, MO, USA).
7
MITF ChIP-qPCR and ChIP-seq Analysis
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For chromatin immunoprecipitation (ChIP) analysis 2×107 501 mel cells were utilized for DNA-protein cross-linking with 1% formaldehyde. For immunoprecipitation, sheared chromatin equaling 1×106 cells was incubated with 5 µg of either goat polyclonal anti-MITF (Santa Cruz, sc-11002X) or normal goat IgG (Santa Cruz, sc-2028) antibody for GTF2H1 promoter analysis. For CDK7 promoter analysis we used a mouse monoclonal anti-MITF (Active Motive, 39789) or a rabbit polyclonal anti-c-MYC (Cell Signaling, #9402) and isotype control mouse IgG1 (Biolegend, 401402) and rabbit IgG (Santa Cruz, sc-2027). Quantitative real-time PCR was applied on a Light Cycler instrument (Roche) to amplify DNA fragments containing E boxes in GTF2H1 or CDK7 promoter. GTF2H1 intron 1 or the CENPH intron 1 were used as negative control loci (primer sequences are available on request). Following ChIP-seq datasets were downloaded from the GEO database and analyzed to obtain extended data on FUBP2/KHSRP and CDK7 loci: Primary melanocytes (GSM1226221), primary melanoma (GSM1484317), COLO829 (GSM1226224), 501 mel (GSM1517751), MCF7 (GSM1006877), P493 (GSM1234501), BL41 (GSM762710), and GM12878 (GSM754334).
8
Melanocyte Protein Expression Analysis
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Melan-a cell lysates were prepared using RIPA buffer supplemented with 1% phenylmethylsulfonyl fluoride and 1X protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysates (40 µg) were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Proteins were measured using antibody-based probes, including anti-tyrosinase, anti-TRP-1, anti-TRP-2, and anti-MITF (Santa Cruz biotechnology, Dallas, TX, USA). GADPH was used as a loading control antibody.
9
Melanosome Protein Expression Analysis
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Cells were lysed in radio-immunoprecipitation assay (RIPA, Sigma, Darmstadt, Germany) containing phenylmethanesulfonyl fluoride (PMSF, final concentration of 1 mM). Lysates were centrifuged at 10,000 rpm for 5 min at 4 °C. Supernatants were discarded and total proteins were obtained. Western blot analysis was performed using the Wes Simple Western system (Protein Simple, New York, USA). Proteins were transferred to membranes at 375 V for 25 min, blocked for 15 min, and probed with indicated primary antibodies for 30 min, followed by labeling with Anti-Mouse Secondary Antibody for 30 min (12–230 kDa Jess or Wes Separation Module, 8 × 25 capillary cartridges, Protein Simple, New York, USA). After 3 h, data were obtained analyzed using Compass software (Protein Simple, New York, USA). Primary antibodies included anti-TYR (1:100 rabbit polyclonal, BBI, Wuhan, China), anti-TYRP1 (1:100 rabbit polyclonal, Abcam, Cambridge, UK), anti-MITF (1:100 rabbit polyclonal, Santa Cruz, CA, USA), and anti-SV40-LT (1:100, mouse monoclonal, Santa Cruz, CA, USA).
10
Melanogenesis Regulation Assay Protocol
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Methyl linoleate, methyl linolenate, α-MSH, L-DOPA, sodium hydroxide (NaOH), mushroom tyrosinase, arbutin, kojic acid, resveratrol, and 6-bromoindirubin-3'-oxime (BIO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies recognizing Akt, p-Akt, GSK3β, p-GSK3β, and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-β-catenin antibody was purchased from BD (Franklin Lakes, NJ, USA), and anti-MITF, anti-tyrosinase, and anti-TRP1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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