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.
Ab20663
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab20663 is a laboratory antibody product manufactured by Abcam. It is intended for use in research applications. The core function of this product is to serve as a tool for scientific investigation, but a more detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab221144, by Abcam (3 mentions)
Ab180753, by Abcam (2 mentions)
Ab154598, by Abcam (2 mentions)
Ab182651, by Abcam (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Ab18075, by Abcam (2 mentions)
P gsk3β ab75814, by Abcam (2 mentions)
Goat anti rabbit igg secondary antibodies ab205718, by Abcam (2 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (2 mentions)
Gene specific primers, by Sangon (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab74073, by Abcam (1 mentions)
Ab 3312, by Abcam (1 mentions)
P0448, by Agilent Technologies (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
P0447, by Agilent Technologies (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab52599, by Abcam (1 mentions)
Ab731, by Abcam (1 mentions)
5 protocols using ab20663
1
Melanogenic Protein Expression Analysis
2
Cryopreservation of Melanoma Cell Lines
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Cryopreservation freezing solution was obtained from ZENOAQ RESOURCE CO., LTD (Fukushima, Japan). DAPI, Live and dead viability/cytotoxicity kit, and MTT assay kit were purchased from Thermo Fisher Scientific (Rockford, IL, USA). MS Collagen (Type 1 atelo-collagen from porcine skin) was obtained from MSBio, Inc. (Gyeonggi, Korea). Antibodies that recognize MITF (ab20663), Mel A (ab731), H2B (ab52599), S100 (ab4066), and tyrosinase (ab738) were purchased from Abcam (Cambridge, UK). Antibodies specific for actin (sc47778) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). A375 melanoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). A375 cells were cultured in DMEM-high glucose with 10% FBS and 1% PS (penicillin-streptomycin).
3
Melanocytic Pathway Regulation Protocol
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Polymyxin B was obtained from USP Reference Standards (lot no. R046V0). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, and 0.25% trypsin were purchased from GIBCO Invitrogen (CA, USA). All chemicals and reagents were purchased from MedChemExpress (NJ, USA) at analytical grade. Gene-specific primers were obtained from Sangon Biotech (Shanghai, China). MITF (ab20663), TYR (ab18075), DCT (ab221144), PI3K (ab154598), p-PI3K (ab182651), GSK3β (ab32391), p-GSK3β (ab75814), β-catenin (ab16051), p-β-catenin (ab75777), p-CREB (ab32096), CREB (ab32515) and β-actin (ab8227) primary antibodies and goat anti-rabbit IgG secondary antibodies (ab205718) were purchased from Abcam (Cambridge, MA, USA).
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
Melanocytic Pathway Regulation Protocol
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Polymyxin B was obtained from USP Reference Standards (lot no. R046V0). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, and 0.25% trypsin were purchased from GIBCO Invitrogen (CA, USA). All chemicals and reagents were purchased from MedChemExpress (NJ, USA) at analytical grade. Gene-specific primers were obtained from Sangon Biotech (Shanghai, China). MITF (ab20663), TYR (ab18075), DCT (ab221144), PI3K (ab154598), p-PI3K (ab182651), GSK3β (ab32391), p-GSK3β (ab75814), β-catenin (ab16051), p-β-catenin (ab75777), p-CREB (ab32096), CREB (ab32515) and β-actin (ab8227) primary antibodies and goat anti-rabbit IgG secondary antibodies (ab205718) were purchased from Abcam (Cambridge, MA, USA).
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