The expressions of melanogenic proteins were determined using the western blotting analysis which was modified from a previous study (16 (link)). Briefly, 40 μig of protein lysate obtained from treated B16 cells was electrophoresed and transferred to polyvinylidene difluoride membrane (Millipore, Germany). The membrane was treated individually with primary antibody including rabbit polyclonal anti-MITF, 1:1000 (ab 20663, Abcam, UK); rabbit polyclonal anti- TYR, 1:1000 (ab 180753, Abcam, UK); mouse monoclonal anti-TRP-1, 1:100 (ab 3312, Abcam, UK); rabbit polyclonal anti-TRP-2, 1:100 (ab 74073, Abcam, UK), and rabbit polyclonal anti-β-actin, 1:1,000 (ab 8227, Abcam, UK) at room temperature for 1 h with agitation before washing with 1× tris-buffered saline and Tween® 20 (TBST), pH 7.2 for three times. The membrane was then treated with secondary antibody including polyclonal goat anti-rabbit IgG-horseradish peroxidase(HRP), 1:1,000 (P0448, Dako, Denmark) and polyclonal goat anti-mouse IgG-HRP, 1:1,000 (P0447, Dako, Denmark) at room temperature for 1 h with gentle agitation. Then, washed the m emb rane with 1× TBST, pH 7.2 for 5 min three times, and incubated with Luminata Forte western HRP substrate (Merck, Germany) for 10 min. The proteins were then visualized using a gel documentation analyzer (ImageQuant LAS 500, UK) and analyzed the densitometry by Image J software.
Ab180753
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab180753 is a monoclonal antibody. It is intended for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab221144, by Abcam (4 mentions)
Ab20663, by Abcam (2 mentions)
Ab7753, by Abcam (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
P0448, by Agilent Technologies (1 mentions)
P0447, by Agilent Technologies (1 mentions)
Ab74073, by Abcam (1 mentions)
Ab 3312, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit or anti goat igg, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Rt qpcr kit, by Takara Bio (1 mentions)
Cdc42, by Santa Cruz Biotechnology (1 mentions)
Rab27a, by Santa Cruz Biotechnology (1 mentions)
Irdye 680lt goat anti mouse, by LI COR (1 mentions)
Radioimmunoprecipitation assay (ripa), by Merck Group (1 mentions)
Ab140606, by Abcam (1 mentions)
9 protocols using ab180753
1
Melanogenic Protein Expression Analysis
2
IgG Peptide Synthesis and Antibody Detection
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The whole Igκ was purchased from Sigma-Aldrich. The human IgGκ (188–203) peptide (sequence KHKVYACEVTHQGLSS) and IgGκ (145–159) peptide (sequence: KVQWKVDNALQSGNS) were produced by Fmoc technology and an Applied Biosystems Synthesizer [30 (link),72 (link)]. Peptide purity (>99%) and sequence were analyzed by reverse phase HPLC purification and mass spectroscopy. The primary antibodies used were human GILT, HLA-DM, CD86, Cat B, Cat D, Cat S, Cat L and β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), tyrosinase (ab180753, Abcam), vimentin (V9, ThermoFisher Scientific, Waltham, MA, USA), and CD11c (N418, ThermoFisher Scientific). HLA-DR (L243) and Ii (Pin 1.1) were obtained from Janice Blum (Indiana University School of Medicine, Indianapolis, IN, USA). The secondary antibodies used were horseradish peroxidase conjugated anti-mouse (Pierce, Rockford, IL, USA), anti-rabbit or anti-goat IgG (Santa Cruz).
3
Melanogenesis Signaling Pathway Analysis
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Cells were lysed with cell lysis buffer (containing 1% PMSF), and protein content was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology, China). The protein was separated by SDS-PAGE, then transferred to a PVDF membrane, and sealed with 5% BSA for 1 h–2 h at room temperature. Anti-TYR (Abcam, ab180753), anti-TRP-1 (Abcam, ab235447), anti-DCT (Abcam, ab221144), anti-MITF (Abcam, ab20663), anti-p-P38/P38 (CST, 4511/8690), anti-p-JNK/JNK (CST, 4668/9252), anti-p-ERK/ERK (CST, 4377/4370), and anti-Actin (Sigma) were incubated at 4°C overnight and then probed with the corresponding second antibody at room temperature for 1 h. The signals were visualized by Tanon 4600SF (Shanghai, China) and determined by quantitative analysis of digital images of gels by using ImageJ version 1.8.0.
4
Protoporphyrin IX and Tyrosinase Melanogenesis Assay
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Protoporphyrin IX (CAS: 553-12-8; purity, >95%) and tyrosinase derived from mushrooms (T128536) were purchased from Aladdin (Shanghai, China). Antibodies against tyrosinase (ab180753, 1:500), MITF (ab20663, 1:2,000), TRP-1 (ab3312, 1:1,000), TRP-2 (ab221144, 1:1,000), and cytokeratin (ab7753, 1:200) were purchased from Abcam (Cambridge, UK). p-CREB (9198S, 1:1,000) and the antibody against CREB (9197S, 1:1000) were obtained from Cell Signaling Technology (MA, USA). LY83583 (sc-200314A), KT5823 (sc-3534B), and antibodies against GP100 (sc-393094, 1:500), KIF5b (sc-133184, 1:500), myosin Va (sc-365986, 1:500), melanophinin (sc-365735, 1: 500), Rab27a (sc-74586, 1: 500), and Cdc42 (sc-8401, 1:500) were obtained from Santa Cruz Biotechnology (CA, USA). RT-qPCR kits were purchased from Takara Biomedical Technology (Beijing, China). The BCA protein assay kit (P0011), cell lysis buffer (P0013), cGMP assay kit, antibody against β-actin (AF0003), donkey anti-rabbit immunoglobulin G (IgG) (Alexa Fluor 555–labeled) (A0453, 1:500), and goat anti-mouse IgG (Alexa Fluor 488–labeled) (A0428, 1:500) were obtained from Beyotime Biotechnology (Shanghai, China).
5
Argan Leaf Saponin and Arbutin Effects on Melanogenesis Proteins
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B16 cells (5 × 104 cells/Petri dishes) were seeded and incubated at 37 °C in an incubator with 5% CO2. After 24 h incubation, the growth medium was replaced with a fresh growth medium with or without 10 μg argan leaf saponin and 100 μM arbutin, and incubated further for 24 h and 48 h. After the specified incubation time, the protein samples were extracted using a RIPA (radio immunoprecipitation assay) lysis buffer (SIGMA; St. Louis, MO, USA) with 0.1% protease inhibitor cocktail (SIGMA; St. Louis, MO, USA) following the manufacturer’s instructions. The protein samples were then loaded into 10% SDS-polyacrylamide gel and subjected to electrophoresis (SDS-PAGE). The proteins in the gel were transferred onto a PVDF membrane and incubated in specific primary antibodies against MITF (ab140606, Abcam, Waltham, MA, USA), TYR (ab180753, Abcam, Waltham, MA, USA), TYRP1 (ab221144, Abcam, Waltham, MA, USA), DCT (ab178676, Abcam, Waltham, MA, USA), and GAPDH (ab181602, ab9485, Abcam, Waltham, MA, USA). Membranes were washed with PBS with Tween-20 (PBST) before incubation with goat anti-mouse IRDye 680LT or goat anti-rabbit IRDye 800CW (LI-COR) secondary antibodies at room temperature.
6
Melanogenesis regulation by MAPK pathways
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J147 (J302241), α-MSH (M118985) and tyrosinase from mushroom (T128536) were obtained from Aladdin (Shanghai, China). We obtained antibodies against Myosin Va (sc-365986), KIF5b (sc-133184), GP100 (sc-393094), Cdc42 (sc-8401), Rab27a (sc-74586), p-JNK (sc-6254), JNK (sc-7345), p-p38 MAPK (sc-166182) and p38 MAPK (sc-398546) from Santa Cruz (CA, USA). The antibodies against MITF (97800), p-MEK (2338), MEK (4694), p-ERK (4370), ERK (4695) were obtained from Cell Signaling Technology (MA, USA). The antibodies against tyrosinase (ab180753), TRP-1 (ab235447), TRP-2 (ab221144), cytokeratin (ab7753) and S100 (ab133519) were obtained from Abcam (Cambridge, UK). p38 inhibitor SB203580 (S1863), ERK inhibitor PD98059 (S1805), BCA protein assay kit (P0012), cell lysis buffer (P0013) and β-actin (AF5001) were obtained from Beyotime (Shanghai, China). RT-qPCR kits (RR036A) were purchased from Takara Biomedical Technology (Beijing, China).
7
Tyrosinase Protein Expression in Adult Skin
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Tissue samples of adult skin were homogenized by sonication in NP-40 lysis buffer (1% NP-40, 150mM sodium chloride, 50mM Tris-HCl pH 8.0, 2mM EDTA, 1mg/mL aprotinin, 1mM PMSF). Protein concentrations were determined using Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Proteins (20 μg total per lane) were separated by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membrane overnight. The membranes were then blocked in 5% bovine serum albumin for 2 hours at room temperature and probed with the antibody against tyrosinase (1:2,000, ab180753, Abcam, Cambridge, UK), followed by incubation with a horseradish peroxidase-coupled anti-rabbit IgG antibody (1:5,000, ab97051, Abcam, Cambridge, UK). Protein bands were visualized via Pierce ECL 2 Western Blotting substrate (#80196, Thermo Scientific, Rockford, IL). Equal loading was verified using an antibody against GAPDH (1:10,000, 5174, Cell Signaling Technology, Danvers, MA). Equal loading was verified using an antibody against alpha-tubulin (1:20,000, ab4074, Abcam, Cambridge, UK).
8
Skin Protein Expression Analysis
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Protein levels of Wnt10b, β-catenin, LEF1, and tyrosinase (Tyr) in the dorsal skin were analyzed by Western blot. Proteins were extracted from skin samples which were lysed by RIPA and PMSF (100:1) buffer at 4°C. After centrifugation at 16000g for 10 min, supernatant was collected. Then proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2 hr and transferred to a polyvinylidene fluoride (PVDF) membrane for 20 min at 20 V. Membranes were incubated separately overnight at 4°C with the primary rabbit antibody: anti-Wnt10b (1:500; ab70816; Abcam, USA), anti-β-catenin (1:1000; #8480; CST, USA), anti-LEF1 (1:1000; ab137872; Abcam, USA), anti-Tyr (1:500; ab180753; Abcam) and anti-GAPDH (1:5000; 10494-1-AP; Proteintech, China) as a control. After washing three times, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (1:5000; ZB2301; ZSGB-BIO, China) at 24°C for 1 hr. The membrane blots were visualized with an enhanced luminol chemiluminescent (ECL) kit (HaiGene, China) after washing three times.
9
Immunofluorescence Staining of Melanoma Cells
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J3.DR4.vec, J3.DR4.GILT cells and melanoma tissues were cultured on glass coverslips (cat# 12-545-80, Fisher Scientific Co., Pittsburgh, PA, USA). Cells and melanoma cultures were washed and fixed with (1:1) acetone/methanol mixture for 10 min at room temperature. Cells were then permeabilized with 0.1% Triton-X 100 for 15 min, blocked with 5% normal serum for 10 min, and incubated with the CD80 and CD86 antibodies at 37 °C for 1 h. Following incubation, cells were washed twice with 1% BSA (cat. No. 2930, OmniPur, EMD, Cincinnati, OH, USA) in PBS, and incubated with Alexa 488-FITC conjugated donkey anti-goat Ig (Santa Cruz) and Alexa 543-rhodamine conjugated goat anti-mouse Ig (Santa Cruz) for 1 h. For the staining of TB#7812 melanoma cultures, primary antibodies including tyrosinase (ab180753, Abcam), vimentin (V9, ThermoFisher Scientific), and CD11c (N418, ThermoFisher Scientific) were used followed by incubation with appropriately matched secondary antibodies. Cells were also counter-stained with DAPI (4′-6-Diamidino-2-phenylindole) for nuclear localization. The slides were mounted in fluorescent mounting medium G (South Biotechnology, Inc., San Francisco, CA, USA), observed with a 63× N.A.1.4 oil immersion objective lens, and analyzed by a Leica TCS SP5 confocal laser scanning microscope using Las-AF software (Leica Lasertechnik, Wetzlar, Germany) [50 (link)].
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