DS was purchased from Sigma-Aldrich (St. Louis, MO, USA). The compound was dissolved in 100% dimethyl sulfoxide (DMSO). A 50 mmol/L stock solution of DS was prepared and stored as small aliquots at –20°C until used. We purchased MTT, DMSO, gelatin, and horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit antibodies from Sigma-Aldrich. Fluorescein isothiocyanate (FITC)-conjugated CD44 and phycoerythrin (PE)-conjugated CD24 antibodies were purchased from BD Biosciences (San Jose, CA, USA). The phospho-specific antibodies anti-p38, anti-AKT, anti-mTOR, and specific antibodies anti-p38, anti-AKT, anti-mTOR, and AKT inhibitor LY294002 were purchased from Cell Signaling Technology (Danvers, MA, USA). HRP-conjugated β-actin, OCT4, proliferation cell nuclear antigen (PCNA), p53, p21, cdc2, cyclin B1, CDK4, cyclin D, CDK2, and cyclin E antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
Anti p38
Manufactured by Cell Signaling Technology
Sourced in United States, China, United Kingdom, Canada
Anti-p38 is a primary antibody that detects the p38 MAPK protein. p38 MAPK is a serine/threonine protein kinase that plays a key role in the cellular response to various types of stress.
Automatically generated - may contain errors
Lab products found in correlation
Anti jnk, by Cell Signaling Technology (154 mentions)
Anti phospho p38, by Cell Signaling Technology (147 mentions)
Anti p p38, by Cell Signaling Technology (127 mentions)
Anti erk, by Cell Signaling Technology (111 mentions)
Anti erk1 2, by Cell Signaling Technology (101 mentions)
Anti phospho jnk, by Cell Signaling Technology (76 mentions)
Anti akt, by Cell Signaling Technology (74 mentions)
Anti p jnk, by Cell Signaling Technology (59 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (51 mentions)
Anti p erk, by Cell Signaling Technology (48 mentions)
Anti phospho erk, by Cell Signaling Technology (47 mentions)
Anti β actin, by Cell Signaling Technology (44 mentions)
Anti gapdh, by Cell Signaling Technology (43 mentions)
Anti p65, by Cell Signaling Technology (36 mentions)
Anti p erk1 2, by Cell Signaling Technology (34 mentions)
Anti phospho akt, by Cell Signaling Technology (34 mentions)
Anti β actin, by Santa Cruz Biotechnology (33 mentions)
Anti iκbα, by Cell Signaling Technology (28 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (27 mentions)
Anti caspase 3, by Cell Signaling Technology (23 mentions)
394 protocols using anti p38
1
Molecular Signaling Pathways in Cancer Cells
2
Western Blot Analysis of Protein Expression
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Cells were lysed with lysis buffer (100 mM Tris-HCl, pH 6.8, 100 mM DTT, 1% SDS, and 10% glycerol) and boiled for 10 mins. Proteins were then separated by 10% SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk in PBS for 45 min before incubation with primary antibodies overnight at 4 °C. Membranes were washed with PBS for three times and incubated with secondary antibody for 1 h at room temperature. Primary antibodies were used as indicated: anti-FLAG M2 (1:10,000 dilution, F1804, Sigma), anti-FBXO16 (1:500 dilution, PA5-66195, Thermo Fisher Scientific, U.S.A.), anti-FBXO16 (1:500 dilution, NBP1-57614, Novus Biologicals, U.S.A.), anti-hnRNPL (1:1000 dilution, sc-32317, Santa Cruz, U.S.A.), anti-SKP1 (1:1000 dilution, sc-5281, Santa Cruz, U.S.A.), anti-CUL1 (1:1000 dilution, sc-17775, Santa Cruz, U.S.A.), anti-HA (1:5,000 dilution, H9658, Sigma), anti-p44/42 MAPK (Erk1/2) (1:1000 dilution, #4695, Cell Signaling Technology), anti-Phospho-p44/42 MAPK (Erk1/2) (1:1000 dilution, #4370, Cell Signaling Technology), anti-p38 (1:1,000 dilution, #8690, Cell Signaling Technology), anti-p38 (1:1000 dilution, #8632, Cell Signaling Technology), and anti-β-actin (1:5000 dilution, sc-8432, Santa cruz, U.S.A.).
3
Characterization of MAPK Pathway Activation
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A subconfluent monolayer of cells was serum-deprived for 4 h in DMEM and then stimulated with 10 mM CaCl2. Cells were harvested with Cell Lysis Buffer® (Cell Signaling, Beverly, MA, USA) according to the manufacturer's instructions. Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (ATTO, Tokyo, Japan). Membranes were blocked for 1 h in 5% (w/v) non-fat dried milk in phosphate-buffered saline with 0.1% (v/v) Tween 20 and incubated with anti-phosphorylated p38Thr180/Tyr182, anti-p38, anti-ERK1/2, or anti-phosphorylated ERK1/2Thr202/Tyr204 (all Cell Signaling) at a 1:1000 dilution at room temperature for 1 h. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) at a 1:2000 dilution at room temperature for 1 h. Proteins were detected using ECL Prime Western blotting detection reagents (GE Healthcare, Madison, WI, USA) according to the manufacturer's instructions and visualized using Molecular Imager® ChemiDoc™ XRS plus (Bio-Rad).
4
Antibody Characterization for Signaling Pathways
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Anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, anti-phospho-MKK3, anti-phospho-MKK4, anti-phospho-ATF2, anti-phospho-MK2, anti-phospho-CREB, anti-phospho-cjun, anti-phospho-IκBɑ, anti-phospho-STAT1, anti-phospho-STAT3, anti-IL-1β, anti-cleaved caspase-3, and anti-phospho-ASK1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ASK1 antibody was provided from Dr. Hidenori Ichijo. Anti-F4/80 antibody was obtained from Serotec. Anti-CD4, anti-caspase-1, and anti-Dclk1 antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-TFF2 antibody was kindly provided by Dr. Sachiyo Nomura. Anti-proton pump and anti-Cdx2 antibodies were obtained from Santa Cruz (Santa Cruz, CA, USA). Anti-BrdU antibody was obtained from Dako (Santa Clara, CA, USA). Anti-actin antibody was purchased from Sigma. Anti-cyclin D1(AB3) was acquired from Invitrogen (Waltham, MA, USA).
5
Western Blot Analysis of Signaling Pathways
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B16-F10 cells (2 × 106 cells per well) were seeded into a 10 cm dish for 24 h, and then cells were treated with different concentrations of PSS (12.5, 25, 50, 100 μg/mL) for 24 h. The medium was removed, and the cells were washed with PBS three times. Cells were then lysed in 200 μL of lysis buffer on ice. The total protein was determined using the Bicinchoninic Acid (BCA) Kit (Solarbio, Beijing, China). Equal amounts of protein in the cell extracts were fractionated by 10% SDS-PAGE, and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with TBST (20 mM Tris-buffered saline and 0.1% Tween) containing 5% nonfat dry milk for 1 h at room temperature, the membranes were incubated for 2 h with monoclonal antibodies, such as anti-MMP-9, anti-MMP-2, anti-E-cadherin, anti-Vimentin, anti-ERK 1/2, anti-p-ERK 1/2, anti-AKT, anti-p-AKT (Ser473), anti-p38, anti-p-p38, anti-p-p38, anti-NF-κB, anti-p-NF-κB, and anti-β-actin, which were purchased from Cell Signaling Technology. The membranes were then washed three times and incubated with HRP-conjugated secondary antibodies (Abcam, Cambridge, Massachusetts, USA). The proteins were then detected using chemiluminescence agents (Amersham ECL, GE Healthcare, Buckinghamshire, UK).
6
Western Blot Analysis of Signaling Proteins
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Samples and pre-stained standard separated on a 4–12% NuPAGE™ (Invitrogen) were transferred onto a piece of Immobilon™ membrane (Millipore, Bedford, MA). The membranes were rinsed with TBS-T (TBS containing 0.05% Tween 20), blocked with 5% nonfat dry milk at room temperature for 1 h, and incubated with appropriately diluted (1:500 ~ 2000) 1st antibodies overnight at 4 °C. The first antibodies were rabbit IgG purchased from Cell Signaling (Beverly, MA), including anti-I-κBα (#9242), anti-GAPDH (#2118), anti-phospho-p44/42 (#9101, Thr202/Tyr204), anti-p44/42 (#9102), anti-phosphorylated p38 (#9211, Thr180/Tyr182), anti-p38 (#9212), anti-phosphorylated JNK (#9251, Thr183/Tyr185), anti-JNK (#9252), anti-phosphorylated p53 (#9284, Ser15), anti-Cdc2 (#77,055), and anti-cyclin B1 (#4138). After washing three times with TBS-T, the membranes were reacted with 1:2000 diluted HRP-conjugated goat anti-rabbit IgG (Cell Signaling, #70,741) for 1 h at room temperature, washed, and developed in SuperSignal® West Dura Extended Duration Substrate (Thermo Fisher Scientific Inc., Hanover Park, IL). The images were collected using the G BOX Chemi systems (Syngene, Frederick, MD).
7
Western Blot Analysis of Cell Extracts
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Western blot analysis of cell extracts was performed as described previously (Ohnishi et al. 2017 (link)). Briefly, cells were washed with phosphate-buffered saline three times then lysed in RIPA buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate, and 1.0% (w/v) NP-40 substitute]. After incubation on ice for 20 min, debris was removed by centrifuging at 2000 g for 10 min at 4 °C. Supernatant proteins (20 μg) were electrophoresed with sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Nonspecific binding was blocked by incubating membranes with Blocking One–P (Nacalai Tesque, Kyoto, Japan) for 20 min at room temperature. Then membranes were incubated overnight with primary antibodies, including anti-phospho-Smad1/5/8 (1:1000), anti-Smad1 (1:1000), anti-phospho-ERK1/2 (1:1000), and anti-ERK1/2 (1:1000), anti-phospho-p38 (1:1000), anti-p38 (1:1000) [all from Cell Signaling Technology, Danvers, MA, USA], at 4 °C and then with secondary antibodies for 45 min at room temperature. Immunoreactive protein bands were visualized using an ECL-peroxidase detection system (Thermo Fisher Scientific) and LAS-4000 (Fujifilm, Tokyo, Japan).
8
Automated Western Blot Analysis of BMMSCs
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Total proteins of BMMSCs were isolated using RIPA (Cell Signaling Technology, USA). The automated western blot was performed using Simple Wes (Protein Simple, USA) following the manufacturer’s protocol. Briefly, 2.5 μg of protein from the cell lysates was added to the standard fluorescent mastermix, then was loaded into corresponding wells of the prefilled Wes assay plate, along with antibody diluent (Protein Simple, USA), anti-MagT1 (Proteintech, USA), anti-DSP (Santa Cruz, USA), anti-DMP-1 (Genetex, USA), anti-ALP (Affinity, USA), anti-ERK (Cell Signaling Technology, USA), anti-p-ERK (Cell Signaling Technology, USA), anti-JNK (Cell Signaling Technology, USA), anti-p-JNK (Cell Signaling Technology, USA), anti-p38 (Cell Signaling Technology, USA), anti-p-p38(Cell Signaling Technology, USA), anti-β-Tublin (Affinity, USA), anti-rabbit secondary antibody (Protein Simple, USA), and Streptavidin-HRP, followed by luminal peroxide mix. The imaging and analysis were done with compass software (Protein Simple).
9
Antibody Characterization for Cellular Signaling
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Antibodies used in this work include: Cetuximab (Creative Diagnostics Cat# AIT-19034, RRID:AB_2489605; Erbitux), anti-Erk1/2 (Cell Signaling Technology Cat# 4695, RRID:AB_390779), anti-phospho-ERK1/2 (T202/Y204) (Cell Signaling Technology Cat# 4370, RRID:AB_2315112), anti-p38 (Cell Signaling Technology Cat# 8690, RRID:AB_10999090), anti-phospho-p38 (T180/Y182) (Cell Signaling Technology Cat# 4511, RRID:AB_2139682), anti-SAPK/JNK (Cell Signaling Technology Cat# 9252, RRID:AB_2250373), anti-phospho-SAPK/JNK (T183/Y185) (Cell Signaling Technology Cat# 4668 P, RRID:AB_10831195), anti-MYC (Cell Signaling Technology Cat# 13987, RRID:AB_2631168), anti-MIZ1 (14300, Cell Signalling), anti-HBD1 (Abcam Cat# ab14425, RRID:AB_301206), anti-actin (Sigma-Aldrich Cat# A2066, RRID:AB_476693), anti-mouse IgG-POX (GE Healthcare Cat# NXA931-1ml, RRID:AB_772209), and anti-rabbit IgG-POX (GAR/IgG(H + L)/PO, Nordic Immunology).
10
Western Blot Analysis of Cardiac Cell Signaling
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The H9c2 cardiac cells were seeded at a concentration of 1 × 104 cells/mL, and incubated at 37°C. The cells with indicated treatments were harvested and dissolved into lysis solution for carrying out western blot testing. Protein concentration was assessed by the bicinchoninic acid protein assay kit. The same amount of cell protein was loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis for electroblotting, and then transferred onto a polyvinylidene fluoride membrane. After being blocked in 5% milk without fat, the membranes were incubated with anti‐p38 (1:1,000 dilution; Cell Signaling Technology, Massachusetts, Boston, USA), anti‐p‐p38 (1:1,000 dilution; Cell Signaling Technology), anti‐ERK1/2 (1:1,000 dilution; Cell Signaling Technology), anti‐p‐ERK1/2 (1:1,000 dilution; Cell Signaling Technology), anti‐p‐JNK, anti‐JNK antibody (1:1,000 dilution; Cell Signaling Technology) or anti‐leptin (1:1,000 dilution; Cell Signaling Technology) at 4°C overnight on a shaker. Then the horseradish peroxidase‐conjugated goat anti‐rabbit as a secondary antibody was used at room temperature for 2 h. The membranes were washed three times with phosphate‐buffered saline with Tween 20, and fluorescence was detected by enhanced chemiluminescence (Bio‐Rad Laboratories Inc., Hercules, California, USA) and exposed to X‐ray film.
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