Aconitine (purity>98%) was purchased from Chengdu Must Bio-Technology Co., Ltd (China) and prepared with dimethyl sulfoxide (DMSO) (Shanghai Solarbio Bioscience and Technology Co., Ltd, China) into the mother liquor with a concentration of 200 mM. Dopamine and its metabolites, namely 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) standards (more than 97% purity), were obtained from Sigma (United States). D1R antagonist SCH23390, D2R agonist sumanirole, and PKA antagonist H-89 were provided by MCE (United States). Anti-TH was acquired from Sigma-Aldrich (United States). Primary antibodies against MAO-B, VMAT2, and D1R were obtained from Abcam (England). Primary antibodies against D2R, DAT, and adenylate cyclase (AC) were purchased from Santa Cruz Biotechnology (United States). PKA, p-PKA, and β-actin rabbit monoclonal antibodies were obtained from Cell Signaling Tech (Danvers, MA, United States). Trizol for RNA extraction was acquired from Invitrogen (United States). All primers for PCR were provided by Biological Engineering Technology Company (Shanghai, China). First-strand cDNA Synthesis kit and SYBR Green Quantitative kits for real-time polymerase Chain Reaction (RT-PCR) were purchased from Tiangen Biotech Co., Ltd. (Beijing, China).
P pka
Manufactured by Cell Signaling Technology
Sourced in United States
P-PKA is a laboratory reagent that detects the phosphorylated form of protein kinase A (PKA). It is used to analyze the activation state of PKA in cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
P creb, by Cell Signaling Technology (9 mentions)
P akt, by Cell Signaling Technology (7 mentions)
P erk, by Cell Signaling Technology (6 mentions)
β actin, by Cell Signaling Technology (5 mentions)
Gapdh, by Santa Cruz Biotechnology (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Trp 2, by Santa Cruz Biotechnology (4 mentions)
Trp 1, by Santa Cruz Biotechnology (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
P erk1 2, by Cell Signaling Technology (3 mentions)
α msh, by Merck Group (3 mentions)
P creb, by Santa Cruz Biotechnology (3 mentions)
L dopa, by Merck Group (3 mentions)
Puromycin, by Merck Group (2 mentions)
P gsk3β, by Cell Signaling Technology (2 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (2 mentions)
Gsk3β, by Cell Signaling Technology (2 mentions)
Arbutin, by Merck Group (2 mentions)
Tyrosinase, by Santa Cruz Biotechnology (2 mentions)
29 protocols using p pka
1
Molecular Mechanisms of Aconitine-Induced Dopaminergic Dysfunction
2
Molecular Profiling of Nuclear Receptors
3
Cellular Assays for Neuroprotection
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6-OHDA, dimethyl sulfoxide (DMSO), paraformaldehyde (PFA), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A lactate dehydrogenase (LDH) kit and cocktail were purchased from Roche Applied Science (Indianapolis, IN, USA). F-12K medium, FBS, HS, penicillin-streptomycin (PS), trypsin-EDTA, and PBS were purchased from Life Technologies (Grand Island, NY, USA). Enhanced chemiluminescence (ECL) solution was obtained from Thermo Fisher Scientific (Rockford, IL, USA). RIPA lysis buffer was bought from Beyotime Biotechnology (Shanghai, China). H-89 was purchased from Selleck Chemicals (Shanghai, China). SYBR® Premix Ex Taq™ II kit was purchased from TaKaRa. (Dalian, China). Antibodies against p-PKA, PKA, p-Akt, Akt, p-CREB, CREB, p-PI3K, PI3K, p-GSK-3β, GSK-3β, β-actin, lamin B1, and HRP-conjugated anti-rabbit IgG were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against Nrf2, PGC-1α, NRF1, and TFAM were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). XF cell culture microplates and XF96 extracellular flux assay kits were purchased from Seahorse Bioscience (North Billerica, MA, USA). All other chemicals of analytical grade were purchased from local sources.
4
Melanogenesis Modulation via Chemical Compounds
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L-3,4-dihydroxyphenylalanine (L-DOPA), α-melanocyte stimulating hormone (α-MSH), and arbutin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM) was obtained from Welgene (DG, KR). Fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Thermo Fisher Scientific (MA, USA). Lysis buffer and 3-(4, 5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) were purchased from Promega (WI, USA). Antibodies specific to MITF, tyrosinase, TRP-1, TRP-2, p38, and GAPDH were purchased from Santa Cruz Biotechnology (CA, USA). Antibodies specific to PKA, CREB, ERK, JNK, p-CREB, p-PKA, p-p38, p-ERK, and p-JNK proteins were obtained from Cell Signaling Technology (MA, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch, Inc. (PA, USA). Western Bright™ ECL reagent was purchased from Advansta, Inc. (CA, USA). All other chemicals were used in analytical reagent grade.
5
Western Blot Analysis of Protein Expression
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The total protein was extracted using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) with protease inhibitor and phosphatase inhibitors (Beyotime, Nanjing, China) and quantified by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Total protein extracts (30 µg) from exosomes, cells, and submandibular gland tissue were separated via 10% SDS‑PAGE (Epizyme, Shanghai, China) and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA) and blocked with 5% non‑fat milk for 1 h at room temperature. After incubation with the primary antibody and the secondary antibody, the target protein was visualized by chemiluminescence using an ECL kit (Beyotime, Nanjing, China). The antibodies used in the Western blot assay are listed as follows: GAPDH (1:10,000, ab181602, Abcam), AQP5 (1:1000, sc-514022, Santa Cruz), CD63 (1:1000, ab134045, Abcam), TSG101 (1:1000, ab125011, Abcam), ALIX (1:1000, ab275377, Abcam), GPER (1:1000, ab260033, Abcam), CREB (1:1000, ab32515, Abcam), p-CREB (1:5000, ab32096, Abcam), PKA (1:1000, #4782, Cell Signaling Technology), p-PKA (1:1000, #4781, Cell Signaling Technology) and goat anti-rabbit IgG H&L (HRP) antibody (1:5000, #31460, Thermo Fisher Scientific).
6
Molecular Signaling in Substance Abuse
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METH (98%) was offered by the Hubei Public Security Bureau. Krill oil was provided by the Aker BioMarine Antarctic Company (Norway). Antibodies against protein kinase A (PKA), phosphorylated protein kinase A (p-PKA), cAMP-response element-binding protein (CREB), phosphorylated CREB (p-CREB), extracellular regulated protein kinase 1/2 (ERK1/2), and phosphorylated ERK1/2 (p-ERK1/2) were bought from Cell Signaling Technology (Danvers, United States). Antibodies against the dopamine D1 receptor (DRD1), dopamine D2 receptor (DRD2), dopamine transporter (DAT), and cleaved caspase-3 were obtained from Absin Bioscience Co., Ltd. (Shanghai, China). Antibodies against GAPDH, horseradish peroxides (HRP)-conjugated goat anti-rabbit antibody and HRP-conjugated goat anti-mouse antibody, protein extraction buffer, protease inhibitors, and phosphatase inhibitors were obtained from Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China). Antibodies against the brain-derived neurotrophic factor (BDNF) were purchased from Santa Cruz Biotechnology (Santa Cruz, United States). All other reagents used were of analytical grade.
7
Western Blot Analysis of Cardiac Proteins
8
Western Blot Analysis of Signaling Proteins
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PAGE was performed using BioRad precast Protein TGX gels. Proteins were transferred to PVDF membranes using a BioRad Trans-Blot Turbo transfer system with the preprogrammed mixed molecular weight setting. Antibodies used for Western blotting purchased from Cell Signaling Technology included:
p-ERK1/2(T202/204)(D13.14.4E), ERK1/2 (137F5), CSK (C74C1), p-ZAP70(Y319) (65E4), ZAP70 (D1C10E), p-P85(Y458), (E3U1H), P85 (19H8), P110 (D1Q7R), p-AKT(S473) (D9E), AKT (C67E7), p-S6 (S235/236)(D57.2.2E), S6 (5G10), STAT3 (124H6), SMAD3 (C67H9), p-PKA(T197) (D45D3) and PKA (D38C6). Antibodies used for Western blotting purchased from Thermo Fisher included: p-CSK(S364) (PA5-40214). Antibodies used for Western blotting purchased from Abcam included: p-STAT3 (Y705), p-SMAD3 (S423,S425) (EP823Y). All primary antibodies utilized were rabbit. An anti-Rabbit IgG-HRP antibody (Cell Signaling Technology 7074) was used with the SuperSignal West Femto chemiluminescent substrate for detection on either a protein simple FluorChem M or a BioRad ChemiDoc system. Densitometry quantitation was performed with the ImageJ software package.
p-ERK1/2(T202/204)(D13.14.4E), ERK1/2 (137F5), CSK (C74C1), p-ZAP70(Y319) (65E4), ZAP70 (D1C10E), p-P85(Y458), (E3U1H), P85 (19H8), P110 (D1Q7R), p-AKT(S473) (D9E), AKT (C67E7), p-S6 (S235/236)(D57.2.2E), S6 (5G10), STAT3 (124H6), SMAD3 (C67H9), p-PKA(T197) (D45D3) and PKA (D38C6). Antibodies used for Western blotting purchased from Thermo Fisher included: p-CSK(S364) (PA5-40214). Antibodies used for Western blotting purchased from Abcam included: p-STAT3 (Y705), p-SMAD3 (S423,S425) (EP823Y). All primary antibodies utilized were rabbit. An anti-Rabbit IgG-HRP antibody (Cell Signaling Technology 7074) was used with the SuperSignal West Femto chemiluminescent substrate for detection on either a protein simple FluorChem M or a BioRad ChemiDoc system. Densitometry quantitation was performed with the ImageJ software package.
9
Western Blot Analysis of Signaling Pathways
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Cells were lysed in a sample buffer containing 2% SDS, 60 mmol/L Tris‐HCl (pH 6.8) and 5% glycerol. Cell lysates were boiled for 5 minutes. Protein concentration was determined using a BCA kit (Beyotime, Shanghai, China), and equal amount of protein was loaded for Western blot analysis as previously described.11 Primary antibodies against ERK1/2, p‐ERK1/2, p38, p‐p38, JNK1/2, p‐JNK1/2, PKCδ, p‐PKCδ, PKA, p‐PKA, MARK2, p‐MAPK, LKB1, p65 and p‐p65, as well as secondary antibodies, were all from Cell Signaling Technology (Boston, MA, USA). Anti‐β‐actin antibody was from Proteintech (Wuhan, China). Blots were developed using enhanced ECL chemiluminescence reagents (Pierce, Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instruction. β‐actin was used as a loading control.
10
Melanocyte Signaling Pathway Analysis
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α-MSH, EGC, H89, SB203580, and TRIzol reagent used in the present study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s Medium (DMEM), fetal bovine serum, penicillin/streptomycin, L-glutamine, and trypsin-EDTA (ethylenediaminetetraacetic acid) were purchased from Hyclone Laboratories (UT, USA). Antibodies including CREB, ERK (extracellular signal-regulated kinases), JNK1 (c-Jun N-terminal kinases 1), JNK2 (c-Jun N-terminal kinases 2), laminin B, MC1R, MITF, p-CREB, TRP-1, TRP-2, and tyrosinase were purchased from Santa Cruz Biotechnology (Dallas city, TX, USA). p38 MAPK, phospho-p38 (p-p38) MAPK, phospho-JNK, PKA, and p-PKA were purchased from Cell Signaling Technology (Beverly, MA, USA). Phospho-ERK and β-actin were purchased from Novus Biologicals (Littleton, CO, USA). The second antibody conjugated with horseradish peroxidase was purchased from Santa Cruz and Sigma-Aldrich.
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