Cytokines (IL-37, IL-19, CXCL1, CXCL2 and CXCL13) were purchased from Cloud-Clone Corp (Wuhan, China), and L-dopa and sodium deoxycholate from Solarbio (Beijing, China). TritonX-100 was purchased from Sigma-Aldrich. Cell Counting Kit-8 (CCK8) and 4% neutral paraformaldehyde were from Biosharp (Hefei, China). Fetal bovine serum (FBS) from Meisen (Zhejiang, China). Dulbecco’s modified Eagle medium (DMEM), penicillin/streptomycin/amphotericin B, penicillin-streptomycin (P/S), and non-essential amino acids (NEAA) were from Gibco (Maryland, USA). Primary antibodies specific for GAPDH (#AP0066, Bioworld), TYR (BS1484, Bioworld), MITF (STJ94134, St. John’s Laboratory), TYRP1 (ab235447, Abcam), DCT (NBP1-56058, Novusbio), PMEL17(#H1219, Santa Cruz and bs-17478R, Bioss) and MLANA (bs-0051R, Bioss and ET1610-47, HUABIO) were purchased as indicated.
Ab235447
Manufactured by Abcam
Sourced in China, United Kingdom, United States
Ab235447 is a lab equipment product offered by Abcam. It is a device used for performing specific laboratory tasks. The core function of this product is to facilitate precise and controlled experimentation within a laboratory setting. No further details about the intended use or features of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Sodium deoxycholate, by Solarbio (2 mentions)
Nbp1 56058, by Novus Biologicals (2 mentions)
L dopa, by Solarbio (2 mentions)
Neutral paraformaldehyde, by Biosharp (2 mentions)
Cxcl1, by Cloud-Clone (1 mentions)
Cxcl2, by Cloud-Clone (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Cell counting kit 8 cck 8, by Biosharp (1 mentions)
Ab27969, by Abcam (1 mentions)
Ab9324, by Abcam (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
Lsm800 w airyscan, by Zeiss (1 mentions)
Ab955, by Abcam (1 mentions)
Anisomycin, by MedChemExpress (1 mentions)
Kojic acid, by Solarbio (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Bioworld Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
10 protocols using ab235447
1
Cytokine-Mediated Melanogenesis Regulation
2
Immunofluorescence Staining of Melanoma and Macrophages
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The cells were washed with 1 × PBS and fixed with 4% paraformaldehyde for 30 min at 25 °C. Next, 0.1% Triton X-100 was added and incubated at 25 °C for 10 min. Thereafter, we added PBS containing 2% bovine serum albumin (BSA) for 30 min for blocking. Anti-TRP1 antibody (ab235447; Abcam) was diluted at a 1/200 ratio in PBS containing 2% BSA for 2 h at 25 °C. Anti-CD68 antibody (ab955; Abcam), anti-TGF-β antibody (ab27969; Abcam), and anti-IL-6 antibody (ab9324; Abcam) were diluted in a 1/200 ratio in PBS containing 2% BSA overnight at 4 °C. Alexa Fluor®594-conjugated goat anti-rabbit IgG (H + L) antibody (A11037, Molecular Probes, Eugene, OR, USA) and Alexa Fluor®488-conjugated goat anti-mouse IgG (H + L) antibody (A11029, Molecular Probes) were diluted in PBS containing 2% BSA and incubated for 1 h at 25 °C. Finally, 4,6-diamidino-2-phenylindole (DAPI; 10236276001; Roche) was applied at 25 °C for 10 min. The cells were washed with PBS and images were obtained using an upright fluorescence microscope (Axio Imager.M2; Carl Zeiss, Oberkochen, Germany) for iMel and confocal laser scanning microscope (LSM800 w/Airyscan; Carl Zeiss) for macrophages.
3
Sitogluside-Mediated Melanogenesis Regulation
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Sitogluside (purity ≥ 98%) was purchased from ChemFaces (#CFN98, 713, ChemFaces). Dimethylsulfoxide (DMSO) was purchased from Sigma-Aldrich. Neutral paraformaldehyde (4%) was purchased from Biosharp (Hefei, China). Kojic acid, the Fontana-Masson Stain Kit, sodium deoxycholate, and L-DOPA were purchased from Solarbio (Beijing, China). Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco (#C11995500BT, Gibco). Fetal bovine serum (FBS) and Cell Counting Kit-8 (CCK8) were purchased from BI (Kibbutz Beit-Haemek, Israel). PMA (12-O-tetradecanoyl phorbol-13-acetate, the ERK/MAPK activator) was purchased from APExBIO (#N2060, APExBIO). Anisomycin (the p38/MAPK activator) was purchased from MedChemExpress (HY-18982, MedChemExpress). Primary antibodies against ras-related protein Rab-27A (RAB27A) (#69295, CST), extracellular signal-regulated kinase (ERK) (#4695, CST), p-ERK (#4370, CST), c-Jun N-terminal kinase JNK (#9252, CST), p-JNK (#4668, CST), p38 (#8690, CST), p-p38 (#4511, CST), cAMP response element-binding protein CREB (#9107, CST), and p-CREB (#9198, CST) were purchased from Cell Signaling Technology. Primary antibodies against TRP1 (ab235447, Abcam), TRP2 (NBP1-56058, Novusbio), MITF (STJ94134, St. John's Laboratory), TYR (BS1484, Bioworld), and GAPDH (#AP0066, Bioworld) were purchased from Abcam, Novusbio, St. John's Laboratory, and Bioworld, respectively.
4
Melanogenic Pathway Analysis in Vitiligo
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Cultured normal human/vitiligo skin tissues were treated with EFE (200 μg/ml for a week) at 37°C and 5% CO2. Skin tissues were washed with PBS three times, fixed in 4% paraformaldehyde, and then dehydrated and embedded in paraffin. 5 μm skin sections of were cut with a microtome and stained using a Masson–Fontana dye kit (Sbjbio, BP-DL371, China) and anti-HMB45 (SANTA CRUZ, sc-59305) according to the manufacturer’s instructions. Photos were recorded by microscope (OLYMPUS, BX43F, United States).
Mice dorsal skins were cut off and fixed in 4% paraformaldehyde, then dehydrated, and embedded in paraffin. 5 μm skin sections were cut with a microtome, stained using a standard hematoxylin/eosin procedure, and anti-TRP-1 (Abcam, ab235447) with the corresponding fluorescent secondary antibodies (Servicebio, GB25303), procedures refer to manufacturer’s instructions (Servicebio, China). Photos were recorded by a microscope (OLYMPUS, BX43F, United States).
Mice dorsal skins were cut off and fixed in 4% paraformaldehyde, then dehydrated, and embedded in paraffin. 5 μm skin sections were cut with a microtome, stained using a standard hematoxylin/eosin procedure, and anti-TRP-1 (Abcam, ab235447) with the corresponding fluorescent secondary antibodies (Servicebio, GB25303), procedures refer to manufacturer’s instructions (Servicebio, China). Photos were recorded by a microscope (OLYMPUS, BX43F, United States).
5
Immunofluorescence and Electron Microscopy Analysis of Melanoma Cells Treated with Ellagic Acid
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Cells were cultured on coverslips in a 12-well plate and treated with EFE (40 μg/ml) for 72 h. Discarding the supernatant, cells were washed with PBS three times, fixed in 4% paraformaldehyde for 10 min and treated with 0.1% Triton for 5 min, and then sealed off with 5% BSA at room temperature for 1 h. Cells were incubated with the primary antibodies, anti-HMB45 (SANTA CRUZ, sc-59305), anti-TRP-1 (Abcam, ab235447), anti-pan cytokeratin (Abcam, ab86734), and FITC-phalloidin (Enzo, 9 A-ALX-350-268-MC01) at 4°C overnight and then incubated with fluorescent secondary antibodies FITC-AffiniPure goat (Yeasen, F2926441) and rhodamine AffiniPure (Yeasen, R1002580) for 1 h at room temperature. Cells were washed with PBS three times and mounted with a mounting medium (with DAPI) (Thermo Fisher, P36935). Photos were recorded by confocal microscope (Leica, SP8).
B16F10 cells were treated with EFE (40 μg/ml) for 72 h and then centrifuged, collected, and fixed in 2.5% glutaraldehyde. The cells were washed with buffer three times for 10 min–20 min, and fixed in 1% osmic acid, dehydrated by gradient ethanol, embedded, and stained. The pictures were recorded by a transmission electron microscope (FEI, Tecnai G2 Spirit BioTWIN).
B16F10 cells were treated with EFE (40 μg/ml) for 72 h and then centrifuged, collected, and fixed in 2.5% glutaraldehyde. The cells were washed with buffer three times for 10 min–20 min, and fixed in 1% osmic acid, dehydrated by gradient ethanol, embedded, and stained. The pictures were recorded by a transmission electron microscope (FEI, Tecnai G2 Spirit BioTWIN).
6
Western Blot Analysis of Melanogenic Proteins
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After the treatment of B16 cells and HaCaT cells, protein extraction was prepared in RIPA buffer (R0010, Solarbio, China), followed by separation on 10% SDS-PAGE gels. Following conventional protocols, these proteins were then transferred from gel to nitrocellulose membranes. 5% BSA was used to block the membrane at room temperature for 1.5 hours. The membrane was incubated with the respective primary antibody overnight at 4°C. Primary antibodies included anti-tyrosinase (abs131593, Absin, China), anti-TRP-1 (ab235447, Abcam, England), anti-TRP-2 (abs131399, Absin, China), anti-MITF (abs100546, Absin, China), and anti-IL-1β (32165, SAB, USA) at a 1 : 1000 dilution. Afterwards, membranes were incubated for 1 hour with 1% skim milk in TBS-T buffer containing horseradish peroxidase-conjugated secondary antibodies (diluted to 1 : 2000). The protein bands were visualized using an ECL system (5200, Tanon, China). The analyzing software (ImageJ) was used to analyze the density of each band. β-Actin and β-tubulin (ab108342, Abcam, England) were used as control for protein loading.
7
Protein Expression Analysis in Ovarian Cancer
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The transfected OVCAR3 and/or SK-OV-3 cells were lysed with RIPA buffer (Beyotime Institute of Biotechnology) to extract total protein. The protein concentration was detected via the BCA Protein Assay kit. Subsequently, a total of 50 µg protein/lane was separated by 10% SDS-PAGE and then transferred onto PVDF membranes. Following blocking with 5% skimmed milk for 2 h at 25˚C, the membranes were incubated overnight at 4˚C with primary antibodies, including anti-TYRP1 (1:1,000; ab235447; Abcam), anti-Bax (1:1,000; ab32503; Abcam), anti-Bcl-2 (1:2,000; ab182858; Abcam) and anti-β-actin (1:1,000; ab265588; Abcam). Thereafter, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; ab97080; Abcam) at 25˚C for 1 h. The proteins bound by their respective antibodies on the immunoblots were measured using an enhanced ECL kit (Thermo Fisher Scientific, Inc.) and quantified using ImageLab software (version 2.3; Bio-Rad Laboratories, Inc.).
8
Western Blot Analysis of Melanogenic Proteins
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The protein was extracted from the cells as described for the tyrosinase activity assay. Proteins were separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with PBS containing 0.1% Tween 20 (PBST) and 3% BSA for 1 h and then incubated with an anti-MITF antibody (1:1000, ab12039; Abcam), anti-tyrosinase antibody (1:500, sc-20035; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-TRP1 antibody (1:1000, ab235447; Abcam) at 4 °C. The next day, membranes were washed 3 times with 0.1% PBST and incubated for 1 h with the respective secondary antibodies. After 4 washes with 0.1% PBST, the protein-of-interest was detected using a Bio-Image Analysis System (Amersham Imager 600; Fuji Photo Film Co., Ltd., Tokyo, Japan).
9
Western Blot Analysis of TYRP1 and GAPDH
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Western blot was performed according to the methods of previous published paper.20 (link) The following primary antibodies were used: TYRP1 (ab235447, 1:1000, Abcam) and GAPDH (60004-1-Ig, 1:8000, PROTEINTECH GROUP). After four washes with Tris buffered saline Tween, the membranes were incubated with Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibodies (SA00001-2, 1:5000, PROTEINTECH GROUP) or goat anti-mouse IgG secondary antibodies (SA00001-1, 1:5000, PROTEINTECH GROUP) and were visualized by MiniChem610 (SAGECREATION, Beijing, China). The intensity of protein bands was quantified with Image J 1.53t software.
10
Notch Signaling Pathway Regulation
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Hexa-D-arginine (D6R) (Cat. No. HY-P1028, 99.57% purity), valproic acid (VPA) (Cat. No. HY-10585, ≥98.0% purity), and levodopa (L-DOPA) (Cat. No. HY-N0304, 99.98% purity) were purchased from MedChemExpress (MCE) (Monmouth Junction, NJ, USA). Antibodies against MITF (12590S, 1:4000), Rab27A (69295S, 1:1000), Myo5a (3402S, 1:1000), FSCN1 (99978S, 1:1000), Notch-1 (4380T, 1:1000), Notch-2 (5732T, 1:1000), Notch-3 (5276T, 1:1000), Jagged-1 (2620T, 1:1000), Jagged-2 (2210T, 1:1000), delta-like ligand 1 (DLL-1) (2588T, 1:1000), delta-like ligand (DLL-4) (2589T, 1:1000), hairy and enhancer of split-1 (Hes-1) (11988S, 1:1000), Cleaved Notch-1 (N1ICD) (4147S, 1:1000), and β-actin (4970S, 1:2000) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody against furin (MA534677, 1:1000) was obtained from Invitrogen (Waltham, MA, USA). Antibody against delta-like ligand 3 (DLL-3) (25535-1-AP, 1:1000) was obtained from proteintech (Rosemont, IL, USA). Finally, antibodies against TYR (ab170905, 1:4000), TYRP-1 (ab235447, 1:8000), and TYRP-2 (ab221144, 1:4000) were obtained from Abcam (Cambridge, UK).
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