For Western blot analysis, the cells were harvested and lysed using RIPA lysis buffer (supplemented with 1× PMSF). Equal protein amounts (20–30 µg) were separated by SDS-PAGE and then blotted onto PVDF membranes. The blots were blocked with 5% non-fat milk for 1 h, probed with primary antibody at 4°C overnight, washed with TBS-Tween-20 three times, and finally incubated with secondary antibody for 1 h at room temperature. Immunoreactive blots were visualized with ECL Plus reagents. The following primary antibodies were purchased from Cell Signaling Technology: YAP (#14074, 1:1000), β-catenin (#8480, 1:1000), E-cadherin (#14472, 1:1000), N-cadherin (#13116, 1:1000), vimentin (#5741, 1:1000), Slug (#9585, 1:1000), P38 (#8690, 1:1000), phosphorylated p38 (pP38) (#4511, 1:1000), ERK (#4695, 1:1000), phosphorylated ERK (pERK) (#9010, 1:1000), c-Jun NH2-terminal kinase (JNK) (#9252, 1:1000), and phosphorylated JNK (pJNK) (#9255, 1:1000).
Phosphorylated p38 p p38
Manufactured by Cell Signaling Technology
Sourced in United States
Phosphorylated-p38 (p-p38) is a lab equipment product that detects the phosphorylated form of the p38 mitogen-activated protein kinase (MAPK). p38 MAPK is a key signaling molecule involved in various cellular processes, including stress response, inflammation, and cell differentiation.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (5 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Total p38 t p38, by Cell Signaling Technology (2 mentions)
P spak, by OriGene (2 mentions)
Phosphorylated erk1 2 p erk1 2, by Cell Signaling Technology (2 mentions)
Ccd camera, by Bio-Rad (2 mentions)
15 lox, by Abcam (2 mentions)
Phosphorylated p65 p p65, by Cell Signaling Technology (2 mentions)
5 lox, by Cayman Chemical (2 mentions)
Cd206, by Abcam (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Osterix, by Abcam (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
15 protocols using phosphorylated p38 p p38
1
Western Blotting Procedure for Protein Analysis
2
Protein Expression Analysis of Cell-Scaffold Constructs
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At the 7th, 14th, and 21th days, cell-scaffold complex was trimmed, then immersed in lysate (RIPA lysis buffer with 1 mM PMSF, Beyotime), and ultrasonically homogenized on ice to extract total protein. Concentration of protein samples was measured using BCA protein assay kit. After being mixed with loading buffer, protein samples were denatured in 95°C water bath for 10 min. Protein bands (30μg per lane) were separated by electrophoresis and transferred on PVDF membranes to be incubated with the following primary antibodies: β-actin (Proteintech), Runt-related transcription factor 2 (Runx-2), Distal-less homeobox 5 (Dlx-5), osterix (Abcam), collagen I (col1, Merck Millipore), Smad1/5/9, phosphorylated-Smad1/5/9 (pSmad1/5/9), serine/threonine kinase (Akt), phosphorylated-Akt (pAkt), p38 mitogen-activated kinase (p38), and phosphorylated-p38 (p-p38, Cell Signaling) at 4°C overnight and corresponding second antibodies under room temperature for 2 hours, developed using ECL gel imaging system. The grey level of protein bands was measured using ImageJ software; relative expression of the above indexes was determined by their grey levels divided by that of β-actin.
3
Nrf2/HO-1 Activation Mitigates Oxidative Stress
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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotics and other tissue culture reagents were purchased from Gibco BRL Co. (Gaithersburg, MD, USA). Lipopolysaccharide (LPS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis, MO, USA). Cobalt protoporphyrin IX (CoPP), Tin protoporphyrin IX (SnPP), PD98059, SB203580 and SP600125 were purchased from Enzo (Enzo Lifesciences, Farmingdale, NY, USA). Primary antibodies, including Heme oxygenase-1 (HO-1) and Nuclear transcription factor erythroid-2 related factor 2 (Nrf2) antibodies, were purchased from Abcam (Abcam Plc, Cambridge, UK). Phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK), phosphorylated JNK (p-JNK), and phosphorylated p38 (p–p38) antibodies and secondary antibodies used for western blot were purchased from Cell Signaling Technology (Cell Signaling Technology Inc., Beverly, MA, USA). All other chemicals were obtained from Sigma Chemical Co. BJ-1201, as a derivative of the aminopyridinol compound, was obtained from Dr. Jeong at Yeungnam University as previously described [16 (link)].
4
Western Blot Analysis of Mouse Skin
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The mouse skin tissues and whole-cell extracts were prepared in RIPA lysis buffer for Western blotting (Cat# P0013, Beyotime, China) and centrifuged at 13700 × g for 15 min at 4°C. After obtained the supernatants, protein concentrations were detected using a Bradford Protein Quantification Kit (Cat# P0010, Beyotime, China). The protein samples were loaded and separated by SDS–PAGE (Cat# 1610183, Bio–Rad, United States) and then transferred to PVDF membranes (Cat# FFP26, Beyotime, China). Membranes were incubated overnight at 4°C with specific primary antibodies. Sequentially, membranes were incubated with secondary antibodies and visualized using a ChemiDoc XRS System (ChemiDoc, Bio–Rad, United States). Primary antibodies used for Western blotting were as follows: stathmin (Cat# 11157-1-AP, Proteintech, United States), PCNA (Cat# 13110S, Cell Signaling Technology, United States), p38 (Cat# 8690, Cell Signaling Technology, United States), phosphorylated p38 (p-P38, Cat# 9211S, Cell Signaling Technology, United States), and β-actin (Cat# 2148S, Cell Signaling Technology, United States).
5
Western Blot Analysis of Cell Signaling
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Specimens of cells and tissue were harvested and incubated for 10 min on ice with lysis buffer. The lysates were then centrifuged at 14,000 RCF for 10 min at 4 °C, and the supernatants were collected. Equal amounts of protein samples were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto a polyvinylidene difluoride membrane (Millipore). Western blot analyses were performed with the relevant antibodies: claudin-18 (diluted 1:200, Thermo Fisher Scientific Inc, IL, USA), p-SPAK (diluted 1:1000, OriGene, MD, USA), phosphorylated-p38 (p-p38) (diluted 1:1000, Cell Signaling Technology, USA), total-p38 (T-p38) (diluted 1:1000, Cell Signaling Technology, USA), beta-actin (diluted 1:1000, Sigma Chemical Company, MO, USA) and GAPDH (diluted 1:1000, Thermo Fisher Scientific Inc, IL, USA).
6
Protein Expression Analysis of Acacetin-Treated Cells
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Cells were treated with acacetin and lysed using protein lysis buffer (Sigma). Equal amounts of protein were separated on 8–10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, United States). The PVDF membranes were blocked, incubated with primary antibodies overnight at 4°C, incubated with secondary antibodies, and the signal was detected with Luminol/Enhancer solution (Millipore). Protein bands were quantitated using the BioSpectrum 600 system (UVP, Upland, CA, United States). The primary antibodies included antibodies that recognized the following proteins: phosphorylated-AMPKα, AMPK, IκBα, phosphorylated-IκBα, p65, and lamin B1 (Santa Cruz, CA, United States); ERK1/2, phosphorylated-ERK1/2 (pERK1/2), JNK, phosphorylated-JNK (pJNK), p38, phosphorylated-p38 (pp38), fatty acid synthase (FAS), fatty acid binding protein (aP2), sirtuin 1 (Sirt1), SREBP-1c, and lipoprotein lipase (LPL) (Cell Signaling Technology, Danvers, MA, United States); acetyl CoA carboxylase-1 (ACC-1), phosphorylated-ACC-1 (pACC-1), adipose triglyceride lipase (ATGL), C/EBPα, C/EBPβ, PPAR-α, PPAR-γ, hormone-sensitive lipase (HSL), phosphorylated-HSL (pHSL) (Abcam, Cambridge, MA, United States); and β-actin (Sigma).
7
Osteoclastogenesis Inhibition Protocol
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Rhaponticin (purity >98%) was purchased from Ruifensi company (Chengdu, China) and dissolved with dimethyl sulfoxide (DMSO), stocking at the concentration of 100 mM in −20°C freezer. Further dilution was achieved through adding phosphate-buffered saline (PBS) to the original stock. The complete cell medium consisted of Alpha modified minimal essential medium, penicillin/streptomycin (1%), and fetal bovine serum (10%), obtained from Thermo Fisher Scientific (Scoresby, Vic, Australia). Recombinant M-CSF and glutathione S-transferase-recombinant RNAKL (GST-rRANKL) were used as previously described (Xu et al., 2000 (link)). The regents for MTS and luciferase assay were purchased from Promega (Madison, WI, United States). The primary antibodies special for NFATc1, c-Fos, CTSK, V-ATPase-d2, integrin β3, extracellular signal regulated kinase (ERK), phosphorylated ERK (p-ERK), P38, phosphorylated P38 (p-P38), and β-actin were obtained from Cell Signaling Technology, Santa Cruz Biotechnology and Abcam companies.
8
PAR4-AP Stimulation in ESCC Cells
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ESCC cells were continuously stimulated with PAR4-AP at a concentration of 100 μM for 1, 2, 6, 12, and 24 h. Then, cells were lysed, and protein was extracted. Protein lysate from each sample was separated electrophoretically in sodium dodecyl sulfate polyacrylamide gel and then transferred to polyvinylidene fluoride (PVDF) membranes. Western blot analyses were performed with anti-DNMT1, HDAC2, p16 (Abcam), PAR4 (Alomone Labs), phosphorylated-ERK1/2 (p-ERK1/2), phosphorylated-p38 (p-p38), and GAPDH (Cell Signaling Technology).
9
Luteolin Modulates Inflammatory Pathways
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Luteolin (purity > 98%; molecular weight, 286.24; chemical formula C15H10O6), LPS, dimethylsulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). Antibodies against NF-κB p65, p38, phosphorylated p38 (p-p38), JNK, phosphorylated JNK (p-JNK), ERK, phosphorylated ERK (p-ERK), Akt and phosphorylated Akt (p-Akt) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against TLR-4 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-β-actin antibody was purchased from Sigma.
10
Protein Expression Analysis in Vascular Cells
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Aliquots of protein extracts derived from THP-1 monocytes, THP-1-derived macrophages, HASMCs, and HUVECs were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then immunoblotted with antibodies raised against the following proteins: CD36, CD68, ACAT-1, ICAM-1, VCAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ABCA1, phosphorylated nuclear factor-κB (p-NF-κB), phosphorylated c-jun N-terminal kinase (p-JNK), α-tubulin, arginase-1 (GeneTex, Irvine, CA, USA), E-selectin, MARCO (Bioss, Woburn, MA, USA), peroxisome proliferator-activated receptor-γ (PPAR-γ; Signalway Antibody, College Park, MD, USA), phosphorylated Akt, phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated p38 (p-p38), Bax (Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (Abcam, Cambridge, UK), cleaved caspase-3 (R&D Systems, Minneapolis, MN, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Acris-OriGene Technologies, Herford, Germany), and β-actin (Sigma, St. Louis, MO, USA) [2 (link), 29 (link)–40 (link)]. Proteins were visualized by enhanced chemiluminescence western blotting detection reagents (GE Healthcare, Amersham, UK).
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