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Ab74073

Manufactured by Abcam
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Ab74073 is a recombinant monoclonal antibody. It is designed for use in various laboratory applications.

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Lab products found in correlation

Ab16667, by Abcam (2 mentions) Alexa fluor 647, by Dianova (2 mentions) Alexa fluor 488, by Dianova (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Ventana benchmark, by Roche (1 mentions) Ab9377, by Abcam (1 mentions) Ab21027, by Abcam (1 mentions) Goat anti lyve1 af2125, by R&D Systems (1 mentions) Rat anti cd68, by BioLegend (1 mentions) Rabbit anti akt 9272, by Cell Signaling Technology (1 mentions) Cy3 conjugated secondary antibody, by Dianova (1 mentions) Rat anti cd45, by BD (1 mentions) Ab20663, by Abcam (1 mentions) Ab180753, 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)

9 protocols using ab74073

1

Immunohistochemical Analysis of Melanoma Markers

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For the mouse experiments: skin samples were fixed with 4% formaldehyde and frozen in OCT compound. For immunohistochemistry, sections were stained as previously described [20] (link). Anti-Dct (rabbit, ab74073, Abcam) was used.
Sections of 2 μm from a tissue TMA were stained with antibodies against Melan A, Hif-1α, TRP-2 and Mib-1. The immunohistochemical staining for all antigens was performed on automated staining systems Melan A, TRP-2/Mib-1 on Ventana Bench Mark, Ventana Medical Systems, Tucson, AZ, USA and Hif-1α on Bond Refine, Vision BioSystems Ltd, Newcastle Upon Tyne, UK. The following antibodies were used: Hif-1α clone mgc3 (Abcam Limited), dilution 1:400; Melan A clone A103 (DAKO A/S), dilution 1:30; Mib-1 clone 30–9 (Ventana-Roche), prediluted.
Lenggenhager D., Curioni-Fontecedro A., Storz M., Shakhova O., Sommer L., Widmer D.S., Seifert B., Moch H., Dummer R, & Mihic-Probst D. (2014). An Aggressive Hypoxia Related Subpopulation of Melanoma Cells is TRP-2 Negative. Translational Oncology, 7(2), 206-212.
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2

Comprehensive Immunohistochemistry Panel

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Primary antibodies: Rabbit anti-cleaved Caspase 3 (9661S, Cell Signaling Technology, Danvers, USA), rabbit anti-Ki-67 (ab16667, Abcam, Cambridge, UK), rat anti-CD31 (DM3614P, Dianova, Hamburg, Germany), goat anti-CD32b (AF1460, R&D Systems, Minneapolis, USA), goat anti-Lyve1 (AF2125, R&D Systems, Minneapolis, USA), rat anti-Endomucin (14–5851–82, Thermo Fisher Scientific, Waltham, USA), rabbit anti-TRP-2 (ab74073, Abcam, Cambridge, UK), rabbit anti-wide spectrum cytokeratin (ab9377, Abcam, Cambridge, UK), goat anti-alpha smooth muscle actin (ab21027, abcam, Cambridge, UK), rat anti-CD45 (550539, BD Biosciences, Franklin Lakes, USA), rat anti-CD68 (137002, BioLegend, San Diego, USA), rabbit anti-Melan A (NBP1-30151, Novus, Minneapolis, USA). For Western blotting: rabbit anti-Akt (9272S, Cell Signaling Technology, Danvers, USA), rabbit anti-phospho-Akt (9271S, Cell Signaling Technology, Danvers, USA). Secondary antibodies: Alexa-Fluor 488, Alexa-Fluor 647 and Cy3-conjugated secondary antibodies were purchased from Dianova (Hamburg, Germany). For Western Blotting a rabbit anti-IgG HRP conjugated antibody was used. (Merck, Darmstadt, Germany).
Dietsch B., Weller C., Sticht C., de la Torre C., Kramer M., Goerdt S., Géraud C, & Wohlfeil S.A. (2023). Hepatic passaging of NRAS-mutant melanoma influences adhesive properties and metastatic pattern. BMC Cancer, 23, 436.
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3

Melanogenic Protein Expression Analysis

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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.
Rodboon T., Sirilun S., Okada S., Kariya R., Chontananarth T, & Suwannalert P. (2020). Modified Riceberry rice extract suppresses melanogenesis-associated cell differentiation through tyrosinase-mediated MITF downregulation on B16 cells and in vivo zebrafish embryos. Research in Pharmaceutical Sciences, 15(5), 491-502.
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4

Comprehensive Antibody Panel for Cell Signaling

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Rabbit anti-CREB serum (244, in-house), rabbit anti-CRTC1 (C71D11, 2587, CST), rabbit anti-CRTC2 serum (6865, in-house), rabbit anti-CRTC3 (C35G4, 2720, CST), rabbit anti-CRTC3-pSer391 (PBL #7408, in-house; Sonntag et al., 2019 (link)), rabbit-anti-hCRTC3 (PBL #7019, in-house; Sonntag et al., 2019 (link)), rabbit anti-mOCA2 (PBL #7431, in-house, this work), rabbit anti-H3AcK27 (ab4729, Abcam), Anti-RNA polymerase II (CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (ab5095)), mouse anti-Tubulin (05-829; EMD Millipore), mouse anti-MITF (clone C5, MAB3747-1 Millipore; MITF ChIP grade ab12039 Abcam), rabbit anti-TYR (ab61284, Abcam), rabbit anti-DCT (ab74073, Abcam), rabbit anti-MLANA (NBP254568H, Novus), rabbit anti-PMEL (ab137078, Abcam) rabbit anti-ERK1/2 (4695, CST), rabbit anti-pERK1/2 (9101, CST), mouse anti-HSP90α/β (SC-13119, Santa Cruz Biotechnology), rabbit anti-Histone H3 (9715, CST).
Ostojić J., Yoon Y.S., Sonntag T., Nguyen B., Vaughan J.M., Shokhirev M, & Montminy M. (2021). Transcriptional co-activator regulates melanocyte differentiation and oncogenesis by integrating cAMP and MAPK/ERK pathways. Cell reports, 35(7), 109136.
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5

Transgene Expression Verification Protocol

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To verify the expression of the transgene, Lenti-X cells were seeded in 10 cm plates and transfected or transduced with plasmids or vectors expressing TRP2 and GFP at 37 °C in atmosphere containing 5% CO2. Thirty-six hours post-transfection or post-transduction supernatants and cells were collected. Equivalent amounts of cells, supernatants, and vector preparations were lysed in lysis buffer (20 mM HEPES, 50 mM NaCl, 10 mM EDTA, 2 mM EGTA, 0.5%, NP-40, 50 mM NaF, 1 mM orthovanadate, 1 mM PMSF, 5 mg/mL of aprotin, and 5 mg/mL of leupeptin). Proteins were separated on 12% SDS polyacrylamide gel and transferred to a nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). B16F10 cells were used as positive controls for expression of TRP2. The filters were saturated overnight with 5% nonfat dry milk (NFDM) in PBST (PBS with 0.1% Tween 20) and then incubated with rabbit anti-TRP2 polyclonal antibody (ab74073, Abcam, Cambridge, UK) for 1 h at room temperature, followed by incubation for 1 h at room temperature with an anti-rabbit HRP-conjugated IgG (Sigma). The immunocomplexes were visualized using chemiluminescence ECL detection system (Luminata Crescendo Western HRP Substrate, Millipore).
Morante V., Borghi M., Farina I., Michelini Z., Grasso F., Gallinaro A., Cecchetti S., Di Virgilio A., Canitano A., Pirillo M.F., Bona R., Cara A, & Negri D. (2021). Integrase-Defective Lentiviral Vector Is an Efficient Vaccine Platform for Cancer Immunotherapy. Viruses, 13(2), 355.
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6

Immunohistochemistry of Hair Follicles

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For whole mount preparation, individual hair follicles were fixed with 4% paraformaldehyde (PFA) (Thermo Fisher Scientific, 50980487) for 30 minutes at room temperature and incubated for 1 hour in a blocking buffer containing 10% goat serum (Sigma Aldrich, G9D23) and 5% BSA (Sigma Aldrich, A3294). The hair follicles were subsequently incubated with diluted primary antibodies overnight at 4°C. TRP-2 (abcam, ab74073; 1:100), Ki67 (abcam, ab15580; 1:100), TRP-1 (abcam, ab186929; 1:100), and γH2AX (abcam, ab81299; 1:100) were used as the diluted primary antibodies. The hair follicles were then incubated with 1:500 diluted secondary antibodies for 1 hour at room temperature. Alexa Fluor 594 (goat anti-rabbit IgG; Thermo Fisher Scientific, A-11012) and Alexa Fluor 488 (goat anti-rabbit IgG; Thermos Fisher Scientific, A-11008) were used as the diluted secondary antibodies. Nuclei was labeled with VECTASHIELD Mounting Medium containing DAPI (Vector Laboratories, H-1200).
In the TUNEL assay, cell death was detected by TUNEL staining (TdT-mediated dUTP-digoxigenin nick end labeling technique) using the “in situ cell death detection kit” (Roche Diagnostics, 11684795910). Images were captured using confocal microscopy (Zeiss Axio Observer Z1 Inverted Phase Contrast Fluorescence microscope). Standard microscopy techniques were used to adjust brightness, contrast, focus, and image capture.
Rachmin I., Lee J.H., Zhang B., Sefton J., Jung I., Lee Y.I., Hsu Y.C, & Fisher D.E. (2021). Stress-associated ectopic differentiation of melanocyte stem cells and ORS amelanotic melanocytes in an ex vivo human hair follicle model. Experimental dermatology, 30(4), 578-587.
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7

Detailed Immunofluorescence Staining Protocol

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Primary antibodies: Rabbit anti-cleaved Caspase 3 (9661S, Cell Signaling Technology, USA), rabbit anti-Ki-67 (ab16667, Abcam, UK), rat anti-CD31 (DM3614P, Dianova, Germany), goat anti-CD32b (AF1460, R&D Systems, USA), rabbit anti-Stabilin-2 peptide 15 antibody [33 (link)], goat anti-Lyve1 (AF2125, R&D Systems, USA), rat anti-Endomucin (14–5851-82, Thermo Fisher Scientific, USA), rabbit anti-TRP-2 (ab74073, Abcam, UK), goat anti-Lama4 (AF3837, R&D Systems, USA), rabbit anti-Desmin (ab15200, Abcam, UK), rabbit anti-Fibronectin (ab23750, Abcam, UK), rabbit anti-Collagen I (R1038, Acris Antibodies, Germany), rabbit anti-Collagen III (R1040, Acris Antibodies, Germany), rabbit anti-Collagen IV (NB120-6586, Novus Biologicals, Germany). Secondary antibodies: Alexa-Fluor 488, Alexa-Fluor 647 and Cy3-conjugated secondary antibodies were purchased from Dianova (Germany).
Wohlfeil S.A., Häfele V., Dietsch B., Weller C., Sticht C., Jauch A.S., Winkler M., Schmid C.D., Irkens A.L., Olsavszky A., Schledzewski K., Reiners-Koch P.S., Goerdt S, & Géraud C. (2022). Angiogenic and molecular diversity determine hepatic melanoma metastasis and response to anti-angiogenic treatment. Journal of Translational Medicine, 20, 62.
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8

Evaluation of SELENOK in Melanoma Cells

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Lysates from primary melanocytes were purchased from Sciencell Research Laboratories (Carlsbad, CA) and NCI-60 validated human melanoma cell lines obtained from the University of Hawaii Cancer Center included SK-Mel2, SK-Mel28, and MALME-3M. These cell lines were cultured in RPMI media with 10% fetal bovine serum and 1% Antibiotic-Antimycotic (all from GIBCO/Thermo Fisher). Primary antibodies for western blots included rabbit monoclonal anti-SELENOK (Epigemonics, Inc., custom antibody), anti-IP3R1 (Santa Cruz Biotechnology, sc-271197), anti-GAPDH (Santa Cruz Biotechnology, sc-47724), anti-Prom1 (Cell Signaling, 58605). Antibodies from Abcam included anti-TRP2 (ab74073), anti-calcineurin A and B (ab137335, ab154650), and anti-calmodulin (ab 105498, respectively). Western blot secondary antibodies were purchased from Li-Cor Technologies and immunofluorescence secondary Alexafluor595 antibody from Thermo. The transgene encoding full-length SELENOK in pcDNA3.1+ (Invitrogen) has been previously described [11 (link)].
Marciel M.P., Khadka V.S., Deng Y., Kilicaslan P., Pham A., Bertino P., Lee K., Chen S., Glibetic N., Hoffmann F.W., Matter M.L, & Hoffmann P.R. (2018). Selenoprotein K deficiency inhibits melanoma by reducing calcium flux required for tumor growth and metastasis. Oncotarget, 9(17), 13407-13422.
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9

Immunofluorescence Characterization of Melanocytes

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Immuno uorescence staining was performed to con rm the passage-6 melanocytes identity. Cultured melanocytes were xed by 4% freshly buffered paraformaldehyde, washed with PBS and incubated with 10% goat serum, followed by incubation with primary antibody mouse anti-Melan-A (Sigma-Aldrich, M6570, St. Louis, MO, USA), S100 (Sigma-Aldrich, S2644 St. Louis, MO, USA), HMB-45 ( ab878, Abcam, Cambridge, UK), and tyrosinase II (ab74073, Abcam, Cambridge, UK) at 4°C, overnight. The cells were washed with PBS and incubated with uorescein isothiocyanate (FITC)-conjugated anti-mouse (F9006) for Melan-A, and HMB-45 and goat anti-rabbit for S100 and tyrosinase II for 60 min at room temperature.
A375 and broblast cells were also stained for Melan-A and S100. Nuclei were counter-stained with 5 µg/ml of 4', 6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) and analyzed by a uorescent microscopy (Nikon, Tokyo, Japan).
Shahbazi A., Zargar S.J., Bajouri A., Mohammadi P., & Aghdami N. (2022). Evaluation of Cultured Melanocytes in Terms of Pigmentation, Genetic Stability and Tumorigenicity in order to Vitiligo patients Transplantation.
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