RIPA buffer (R0010; Beijing Solarbio) was used to prepare cell lysates. After the protein was quantified and denatured, the lysate (40 µg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane. The membranes were incubated with the following primary antibodies: anti‐OPN5 (GTX100173; GeneTex, Irvine, CA, USA), anti‐TYR (GTX16389, GeneTex), anti‐TRP1 (ab235446; Abcam), anti‐TRP2 (ab221144; Abcam), anti‐MITF (ab12039; Abcam), anti‐phospho‐MITF (ab59201; Abcam), anti‐PKC (AF6197; Affinity Biosciences, Cincinatti, OH, USA), anti‐phospho‐PKC (AF3197; Affinity), anti‐phospho‐CAMKII (MD1677‐100; MDL Medical Discovery Leader Biotech Co., Ltd, Beijing, China; mdlbiotech.com), anti‐CAMKII (MD2007‐100; MDL), anti‐actin (MD409‐020), anti‐p38 MAPK (MD2025‐100; MDL), anti‐phospho‐p38 MAPK (MD2084‐100; MDL), anti‐ERK1/2 (MD1853‐100), anti‐phospho‐ERK1/2 (MD1412‐100; MDL), anti‐JNK (MD1929‐20; MDL) and anti‐phospho‐JNK (MD1483‐20; MDL) for specific protein detection, and then incubated with horseradish peroxidase‐conjugated secondary antibodies. Specific bands were visualized using a chemiluminescent reaction (electrogenerated chemiluminescence) (E003‐50; 7Seabiotech, Shanghai, China; 7seapharmatech.com).
Ab221144
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab221144 is a laboratory product offered by Abcam. It is a core piece of equipment designed for specific scientific applications. The product's core function is to facilitate essential research activities, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab180753, by Abcam (4 mentions)
Ab20663, by Abcam (3 mentions)
Ab140606, by Abcam (3 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)
Ab7753, by Abcam (2 mentions)
Ab178676, by Abcam (2 mentions)
Ab170905, by Abcam (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Ab12039, by Abcam (1 mentions)
Gtx16389, by GeneTex (1 mentions)
Af6197, by Affinity Biosciences (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Anti actin, by Merck Group (1 mentions)
10 protocols using ab221144
1
Western Blot Analysis of Melanogenic Proteins
2
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).
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
Molecular Signaling Analysis in Cells
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After treatment with various compounds (0.2% DMSO served as a control), the cells were lysed with cell lysis buffer. The antibodies used were antibodies against Akt (9272S; CST, Danvers, USA), pAktSer473 (4060S; CST), Met (ab216330; Abcam, Cambridge, UK), β-catenin (8480S; CST), non-phospho (active) β-catenin (Ser45) (19807S; CST), non-phospho (active) β-catenin (Ser33/37/Thr41) (8814S; CST), MiTF (ab140606; Abcam), TRP2/DCT (ab221144; Abcam), and β-actin (9272S; CST), and the target bands were detected with a chemiluminescence system (Clinx Science Instruments, Shanghai, China).
6
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.
7
Protein Expression Analysis in Exosomes
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Cells were lysed in RIPA buffer (P0013C, Beyotime, Shanghai, China) with a protease inhibitor cocktail (P8340, Sigma‐Aldrich). The total proteins of lysates or exosomes were separated by SDS polyacrylamide gels and transferred onto PVDF membranes (Millipore, USA). Then, these membranes were blocked with 5% BSA for one hour at room temperature and incubated with primary antibodies overnight at 4°C. Afterwards, the secondary antibodies were added. The blots were observed using the Azure Biosystems C300. The antibodies are listed, as follows: rabbit anti‐Tyrosinase (ab170905; Abcam, Cambridge, MA, USA), rabbit anti‐TRP1 (ab178676, Abcam), rabbit anti‐TRP2/DCT (ab221144, Abcam), rabbit anti‐MITF (ab140606, Abcam), rabbit anti‐β‐catenin (51067‐2‐AP; Proteintech, Wuhan, China), rabbit anti‐SOX1 (20744‐1‐AP, Proteintech), rabbit anti‐CD63 (25682‐1‐AP, Proteintech), rabbit anti‐TSG101 (14497‐1‐AP, Proteintech), Lamin B1 antibody (12987‐1‐AP, Proteintech) and β‐actin (no.3700, Cell Signaling Technology, Danvers, MA, USA).
8
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).
9
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).
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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