Protein samples from cultured cells were prepared by direct lysis in 5× SDS sample buffer, followed by heating at 95 °C for 5 min. Samples were resolved on 8–12% SDS–polyacrylamide gels followed by transfer to PVDF membranes. Membranes were blocked in 5% (w/v) dried milk in Tris-buffered saline/Tween (TBST) for 1 h at room temperature, after which they were incubated with primary antibody, followed by the correct horseradish-peroxidase-conjugated secondary antibody. Blots were developed using Western Bright ECL substrate (Advansta) or Immobilon Western chemiluminescent substrate (Millipore) using a Bio-Rad Gel Doc. Antibodies used were anti-HIF-1α (catalogue no. 14179), anti-IκB-α (9242), anti-phospho-NF-κB-p65 (3033), anti-NF-κB-p65 (8242), anti-phospho-p38 MAPK (9211), anti-p38 MAPK (catalogue no. 9212), anti-phospho-ERK (9101), anti-ERK (4695), anti-phospho-p70-S6K (catalogue no. 9205), anti-p70-S6k (9202), all purchased from Cell Signaling Technology, anti-IL-1β (R&D Systems, AF401-NA), anti-β-actin (Sigma-Aldrich, AC-74), anti-FLAG (Sigma-Aldrich, F1804), L-2HGDH (Antibody Genie, CAB7996), and D-2HGDH (Antibody Genie, CAB16213). anti-HIF-1α from Novus (catalogue no. NB100-449) was used for human blots. Secondary antibodies used were horseradish-peroxidase-conjugated anti-mouse IgG, anti-goat IgG and anti-rabbit IgG (Jackson ImmunoResearch).
Anti phospho p38 mapk
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-p38 MAPK is a laboratory product that detects the phosphorylated form of p38 mitogen-activated protein kinase (MAPK). It is used to monitor the activation status of p38 MAPK, which is a key signaling molecule involved in various cellular processes.
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Lab products found in correlation
Anti p38 mapk, by Cell Signaling Technology (60 mentions)
Anti phospho jnk, by Cell Signaling Technology (25 mentions)
Anti jnk, by Cell Signaling Technology (25 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (21 mentions)
Anti erk1 2, by Cell Signaling Technology (21 mentions)
Anti phospho akt, by Cell Signaling Technology (15 mentions)
Anti phospho sapk jnk, by Cell Signaling Technology (14 mentions)
Anti phospho erk, by Cell Signaling Technology (11 mentions)
Anti β actin, by Merck Group (11 mentions)
Anti akt, by Cell Signaling Technology (10 mentions)
Anti phospho c jun, by Cell Signaling Technology (9 mentions)
Anti nf κb p65, by Cell Signaling Technology (9 mentions)
Anti β actin, by Cell Signaling Technology (9 mentions)
Anti iκbα, by Cell Signaling Technology (8 mentions)
Anti β actin, by Santa Cruz Biotechnology (8 mentions)
Anti erk, by Cell Signaling Technology (7 mentions)
Anti gapdh, by Cell Signaling Technology (7 mentions)
Anti phospho iκbα, by Cell Signaling Technology (7 mentions)
Anti phospho nf κb p65, by Cell Signaling Technology (6 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (6 mentions)
111 protocols using anti phospho p38 mapk
1
Western Blot Analysis of Cellular Signaling
2
Detecting Phospho-Mpk1 in H. polymorpha
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To detect the expression of phospho-Mpk1, the H. polymorpha strains were grown to an OD600 of 1.0 in YPD medium at 37°C, and then one-half of the culture was harvested, while the second half was treated with 0.05% SDS, 2 μg/mL caspofungin (CAS), 2.5 μg/mL tunicamycin (TM), 0.2 mg/mL calcofluor white (CFW), 20 mM caffeine, 10 mg/mL Congo red (CR), or 0.5 M sodium chloride for 2 hr. The cells were harvested and lysed by vortexing with glass beads (425–600 μm in diameter, Sigma) in phosphatase inhibitor lysis buffer [41 (link)]. Supernatants were separated by centrifugation at 2,500 × g for 5 min at 4°C and analyzed with 10% SDS-PAGE. For detection of phosphorylated Mpk1 and phospho-Hog1 proteins, the anti-phospho-p44/p42 MAPK and anti-phospho-p38 MAPK antibodies (Cell Signaling Technology) were used, respectively, and anti-ScHog1 antibody (Santa Cruz Biotechnology) was used to detect Hog1 protein. The anti-beta actin antibody (Abcam) was used as a loading control.
3
Inhibition of RAGE-Mediated Inflammation
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Dulbecco’s modified Eagle medium and penicillin–streptomycin solution were purchased from GIBCO (Grand Island, NY, USA). Fetal bovine serum was provided by Hyclone (Logan, UT, USA). Monoclonal rabbit antibodies, including anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK, anti-p65, anti-phospho-p65, NLRP3, and cleaved caspase-1 were obtained from Cell Signaling Technology (Boston, MA, USA). Receptor for AGE (RAGE) monoclonal rabbit antibody was purchased from AbCam (Cambridge, MA, USA). HRP-marked anti-β-actin antibody was supplied by Biorad (San Diego, CA, USA). 4′-Methoxyresveratrol (3,5-dyhydroxy-4′-methoxylstilbene) was purchased from Great Forest Biomedical (Hangzhou, China). BSA was obtained from ABCONE (Shanghai, China). KI and acetic acid were obtained from Sangon Biotech (Shanghai, China). Methylglyoxal, 2’,7’-dichlorodihydrofluorescein diacetate and other reagents were purchased from Sigma (St. Louis, MO, USA).
4
Investigating Inflammatory Signaling Pathways
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LPS (Escherichia coli, O127:B8) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mas receptor antagonist A779 was obtained from AbBiotech (San Diego, CA, USA). ACE2 inhibitor MLN-4760 was a product from EMD Millipore (Darmstadt, Germany). SB203580 (a specific inhibitor of p38 MAPK), PD98059 (a specific inhibitor of ERK1/2) and SP600125 (a specific inhibitor of JNK) were purchased from Santa Cruz Biotechnology (Delaware, CA, USA). Rabbit anti-ACE2, anti-p50, anti-p65, and mouse anti-phospho-p50, anti-phospho-p65 and anti-IκBα antibodies were procured from Santa Cruz Biotechnology. Rabbit anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-/JNK, anti-phospho-JNK, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, and horse anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). TNF-α and IL-1β kits were purchased from Invitrogen (Eugene, OR, USA). AngII and Ang-(1-7) enzyme-linked immunosorbent assay (ELISA) kits were from Kamiya Biomedical (Seattle, WA, USA).
5
Western Blot Analysis of Signaling Proteins
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Cells were lysed in RIPA buffer. Total proteins (50–150 μg) were resolved under reducing conditions by 7–10% SDS-PAGE and transferred to Immobilon-FL membranes (Merck Millipore, Billerica, MA, USA). Membranes were blocked in TBS containing 0.1% Tween 20 (TBS-T) and 5% milk for 1 hr at 25°C and then incubated overnight at 4°C with the following primary antibodies: anti-phospho-p44/42 MAPK (Thr202/Tyr204), anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-p38 MAPK (Cell Signaling Technology, Inc., Danvers, MA, USA), anti-phospho-ERα (Ser118) (Merck Millipore), anti-ERK2, anti-Bek (C-17), anti-ERα, anti-lamin B, anti-E-cadherin (Santa Cruz Biotechnology) and anti-tubulin (Sigma-Aldrich). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma-Aldrich) for 1 hr at 25°C. Bound antibody was detected by enhanced chemiluminescence detection reagents (Pierce Biotechnology Inc., Rockford, IL, USA), according to manufacturer's instructions. Tubulin served to estimate the protein equal loading. Densitometric analysis was performed with Quantity One Program (Bio-Rad Laboratories S.r.l., Segrate, MI, Italy).
6
NMDA-Induced Chemical LTP in Mouse CA1
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For western immunoblotting, we injected flag-p110γ or tdTomato expressing AAV, together with GFP expressing AAV in the CA1 region of 3 weeks-old C57BL/6 male mice. 2 weeks after injection, CA1 regions expressing green fluorescence were dissected and recovered in ACSF maintained at 32°C for 2 h. CA1 mini slices were treated with NMDA (20 μM) for 3 min to induce chemical LTP and immediately snap-frozen in liquid nitrogen. Slices were homogenized using a dounce homogenizer with lysis buffer (10 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, pH 7.5) containing both protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). Samples were analyzed by western blotting using the following antibodies: anti-phospho-p38 MAPK (Cell Signaling) and anit-p38 MAPK (Cell Signaling).
7
Cardamonin Inhibits NF-κB Signaling
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Cardamonin was purchased from Sigma-Aldrich (St Louis, MO, USA). BAY 11-7082 and MG132 and NAC were purchased from Beyotime Biotechnology (Haimen, China). TNF-α, SP600125, SB203580 were from PeproTech Inc. (Rocky Hill, NJ, USA). Cell Counting Kit-8 was from Dojindo Molecular Technologies (Kumamoto, Japan). Anti-NF-κB p65, anti-phospho-p65 anti-CDK4, anti-p21, anti-PARP, anti-JNK, anti-p38 MAPK, anti-phospho-JNK and anti-phospho-p38 MAPK were purchased from Cell Signaling Technology (Danvers, MA, USA).
8
Antibody Sources for Immunoblotting
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A monoclonal antibody against E-selectin (clone 7A9) was obtained from the American Type Culture Collection; anti-E-selectin (A-10: sc-137203) and anti-NF-kB p65 (C-20: sc-372) antibodies were obtained from Santa Cruz Biotechnology; anti-phospho-NF-κB p65 Ser 536 (#3031), anti-SAPK/JNK (#9252), anti-p38 MAPK (#9212), anti-phospho-SAPK/JNK (#9251), and anti-phospho-p38 MAPK (#4511) antibodies were obtained from Cell Signaling Technology; anti-lamin A/C (SAB4200236) and anti-actin (A5060) antibodies were obtained from Sigma-Aldrich; an anti-α-tubulin antibody (PM054-7) was obtained from Medical & Biological Laboratories; an anti-acetyl-histone H3 antibody (06-599) was obtained from Millipore; an Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (A11017) was obtained from Life Technologies; horseradish peroxidase (HRP)-linked anti-mouse (NA931V) and anti-rabbit (NA9340V) secondary antibodies were obtained from GE Healthcare.
9
Immunoblotting for cell signaling proteins
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Whole-cell [29 (link)] and cytosol and light membrane (secretory vesicles plus plasma membrane) fraction [33 (link)] proteins were obtained, solubilized in Laemmli sample buffer, and then subjected to immunoblot by standard procedures using the following antibodies: 1:1,000 anti-phospho-CREB (#9198), 1:1,000 anti-phospho-p38 MAPK (#9211), and 1:1,000 anti-phospho-ERK (#9106) from Cell Signaling; 1:200 anti-GPER1 (sc-48525-R), 1:1,000 anti-PKA (sc-903), and 1:1,000 anti-IκBα (sc-371) from Santa Cruz Biotechnology; 1:2,000 monoclonal antibody anti-β-tubulin (#T5293) from Sigma-Aldrich; and anti-GP91-phox from Abcam (#ab139371). Blotted proteins were detected and quantified using the Odyssey infrared imaging system (LI-COR Biosciences).
10
Quantitative Western Blot Analysis
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Western blot were performed as previously described (Etna et al., 2015 (link)). Briefly, 25 μg of total protein extracts were separated on 10% SDS-PAGE gel and blotted onto nitrocellulose membranes (Millipore). Blots were incubated with rabbit polyclonal anti-phospho-p38 MAPK (Thr180/Tyr182) (p-p38), rabbit polyclonal anti-phospho-p44/42 MAPK (Thr202/Tyr204) (p-p44/42) and rabbit polyclonal anti-phospho-NF-kB p65 (Ser536) (p-p65) Abs (Cell Signaling Technology, Danvers, MA). Detection was achieved using anti-rabbit horseradish peroxidase-conjugate secondary Ab (Santa Cruz Biotechnology), and visualized with Enhanced Chemiluminescence plus kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). A ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) instrument and ImageLab software (Bio-Rad) were used to reveal and analyze the chemiluminescence signal. For loading control, β-actin levels were quantified by using a mouse anti-β-actin Ab (Sigma Aldrich). Protein amount was normalized to the actin level by ImageLab software. Fold changes of each analyzed protein were calculated by dividing values obtained in infected conditions by those of the uninfected counterpart.
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