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).
Anti phosphorylated p38
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phosphorylated p38 is a lab equipment product from Cell Signaling Technology. It is an antibody that specifically recognizes the phosphorylated form of the p38 mitogen-activated protein kinase (MAPK) protein. The core function of this product is to detect and measure the activation of the p38 MAPK signaling pathway in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti p38, by Cell Signaling Technology (13 mentions)
Anti phosphorylated jnk, by Cell Signaling Technology (7 mentions)
Anti phosphorylated erk, by Cell Signaling Technology (6 mentions)
Anti jnk, by Cell Signaling Technology (6 mentions)
Anti iκbα, by Cell Signaling Technology (5 mentions)
Anti phosphorylated erk1 2, by Cell Signaling Technology (4 mentions)
Anti erk, by Cell Signaling Technology (4 mentions)
Anti erk1 2, by Cell Signaling Technology (4 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti phosphorylated iκbα, by Cell Signaling Technology (3 mentions)
Anti phosphorylated p53, by Cell Signaling Technology (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti bcl 2, by Cell Signaling Technology (2 mentions)
Anti phosphorylated akt, by Cell Signaling Technology (2 mentions)
Anti phosphorylated ampkα, by Cell Signaling Technology (2 mentions)
Anti total p38, by Cell Signaling Technology (2 mentions)
Anti nlrp3, by Cell Signaling Technology (2 mentions)
Anti phosphorylated stat3, by Cell Signaling Technology (2 mentions)
Anti p akt, by Cell Signaling Technology (2 mentions)
Anti caspase 9, by Cell Signaling Technology (2 mentions)
23 protocols using anti phosphorylated p38
1
Western Blot Analysis of Signaling Proteins
2
Western Blotting Protein Analysis
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Western blotting was performed as described previously (6 (link)). Cells were lysed using lysis buffer and the concentration was analyzed by the BCA assay (Beyotime Biotechnology). Whole-cell lysates were separated by SDS-PAGE, transferred to a PVDF membrane (Millipore, Shanghai, China), and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST). The primary antibodies used in the study included anti-AKT (1:1,000, Affinity Biosciences, Changzhou, China), anti-p-AKT (1:1,000, Cell Signaling Technology, Shanghai, China), anti-p38 MAP kinase (1:1,000, Affinity Biosciences), anti-phosphorylated p38 (1:1,000, Cell Signaling Technology), and anti-β-actin (1:2,000, Sangon, Shanghai, China). HRP-conjugated species-specific secondary antibodies were purchased from Beyotime Biotechnology. Signals were generated by enhanced chemiluminescence (Biosharp, Hefei, China) and captured by the Odyssey Fc system (LI-COR Biosciences, Lincoln, NE).
3
Western Blot Analysis of Cell Signaling Proteins
4
Antibody Validation for Neurodegenerative Research
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The following commercially available antibodies were purchased: anti-Aβ oligomer (Millipore, Temecula, CA, #AB9234); anti-GAPDH (Millipore, #MAB374); anti-tyrosine hydroxylase (TH) (Millipore, # MAB318); anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA, USA, #9661); anti-p38 (Cell Signaling Technology, #9212); anti-phosphorylated p38 (Cell Signaling Technology, #9211); GAPDH (Chemicon, Temecula, CA, USA, # MAB374).
5
Immunoblotting Analysis of Apoptosis Signaling
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Anti-PARP, anti-β-actin, anti-caspase-3, anti-caspase-9, VDAC, anti-phosphorylated ERK1/2, anti-phosphorylated p38, anti-phosphorylated JNK, anti-phosphorylated IκBα, anti-ERK1/2, anti-p38, anti-JNK, anti-Bcl-2, and anti-IκBα were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). α-Tubulin, anti-Bax (6A7) and His-probe were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against cytochrome c (for immunofluorescence, clone 6H2.B4; for western blot analysis, clone 7H8.2C12) were acquired from BD Pharmingen (San Diego, CA, USA), and the anti-cytochrome oxidase subunit IV (COX IV) antibody was purchased from Abcam (Cambridge, UK). Dichlorodihydrofluorescein diacetate (H2DCFDA), MitoSOX, DAPI, and DiOC6 were obtained from Molecular Probes (Eugene, OR, USA) and z-VAD-fmk, N-acetyl-cysteine (NAC), horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG, and specific inhibitors of ERK (U0126), p38 mitogen-activated protein kinase (MAPK; SB203580), JNK (SP600125), and nuclear factor-κB (NF-κB; BAY 11-7082) were purchased from Calbiochem (San Diego, CA, USA). Anti-F4/80 and anti-CD11b were performed as recommended by the manufacturer (eBioscience, San Diego, CA, USA).
6
Immunoblotting Analysis of Signaling Pathways
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Stimulation, extraction, SDS–PAGE, and immunoblotting were performed as previously described (Martin et al, 2014 ; Izawa et al, 2017 ). The following antibodies were used for immunoblotting: antiphosphorylated tyrosine (4G10; dilution 1:1,000), antiphosphorylated PLC‐γ1 (#2821S; dilution 1:1,000), anti‐PLC‐γ1 (#2822S; dilution 1:1,000), antiphosphorylated ERK 1/2 (#4376S; dilution 1:1,000), anti‐ERK 1/2 (#4695S; dilution 1:1,000), antiphosphorylated P38 (#4511S; dilution 1:1,000), antiphosphorylated AKT (serine 473, 4058S; dilution 1:1,000), and anti‐Ku70 (4103S; dilution 1:1,000) purchased from Cell Signaling Technology; anti‐CTPS1 (EPR8086B; dilution 1:1,000) purchased from Abcam; and anti‐actin (A2066; dilution 1:1,000) purchased from Thermo Fischer Scientific; anti‐RASGRP1 antibodies from Merck Millipore (MABS146, RASGRP1 epitope recognized unknown; dilution 1:1,000), from Thermo Fischer Scientific (PA5‐25750, raised against 495–521 amino acids of RASGRP1; dilution 1:1,000), and from Abcam (EPR9609, raised against the N‐terminal part of RASGRP1; dilution 1:1,000). Membranes were then washed and incubated with anti‐mouse or anti‐rabbit HRP‐conjugated antibodies from Cell Signaling (dilution 1:10,000). Pierce ECL Western blotting substrate was used for detection.
7
Liver Protein Analysis by Western Blot
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Protein isolation from liver tissue and Western blot analysis were performed as previously described (Dou et al., 2018 (link)). The following antibodies were used: anti-phosphorylated-AMPKα (Thr172), anti-AMPKα, anti-PPARα, anti-CPT-1, anti-acetyl-CoA-carboxylase (ACC), anti-phosphorylated-ACC, anti-SREBP-1c, anti-fatty acid synthase (FAS), anti-DGAT-2, anti-P53, anti-Bcl2, anti-Bax, anti-TLR4, anti-JNK, anti-phosphorylated-JNK, anti-phosphorylated-P38, anti-P38, anti-phosphorylated-ERK, anti-ERK, anti-cleaved-caspase-3, and anti-GAPDH (Cell Signalling Technology, Danvers, MA, United States).
8
Western Blot Analysis of Cellular Signaling
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The cells were lysed on ice using RIPA buffer for 30 minutes by intermittent vortexing and incubation on ice during this period. Protein concentration of whole cell lysates was measured using the Bradford assay (Bio‐Rad). Whole cell lysates (20 μg) for each sample was separated on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. The separated proteins were transferred onto a polyvinylidene difluoride membrane followed by blocking at room temperature using 5% non‐fat milk diluted in 0.1% TRIS‐buffered saline‐Tween‐20 for an hour. This was followed by an overnight incubation with either anti‐TonEBP (Abcam), anti‐VDR (Santa Cruz), anti‐total p38 (Cell Signaling Technology, Danvers, MA), anti‐phosphorylated p38 (Cell Signaling Technology), anti‐CFTR (Abcam), anti‐tubulin (Cell Signaling Technology), or anti‐beta actin (Santa Cruz) primary antibodies at 4°C. The membranes were washed and incubated with the relevant secondary antibodies conjugated with HRP (anti‐mouse and anti‐rabbit; BosterBio, Pleasanton, CA) for an hour at room temperature. The membranes were washed and incubated with Clarity ECL Western blotting substrate (Bio‐Rad) and resulting chemiluminescence was imaged using Image quant (GE Image Quant LAS 500; GE Healthcare, Chicago, IL). Densitometry analysis was done using ImageJ software (version 6, NIH, Bethesda, MD).
9
Modulating T-cell Activation and Apoptosis
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Antibodies against human CD3 and CD28 for the stimulation of T cells were purchased from BioXcell (West Lebanon, NH, USA). Human TNFα recombinant protein was obtained from PeproTech EC Ltd. (London, UK). TRIZOL reagent for RNA isolation, MTT powder (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), and radioimmunoprecipitation assay (RIPA) buffer for cell lysis were provided by Sigma Chemical Co. (St. Louis, MO, USA). Staining reagents for AnnexinV (green) and caspase3/7 (red) were purchased from Essen Bio (Ann Arbor, MI, USA). SYBR Premix Ex Taq was obtained from TaKaRa (Shiga, Japan). Anti-p65, anti-LaminB, anti-IκBα, anti-phosphorylated IκBα, anti-phosphorylated ERK, anti-phosphorylated p38, anti-p38, anti-phosphorylated JNK, and anti-JNK were provided by Cell Signaling Technology (Danvers, MA, USA). Antibodies against actin and ERK were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). ECL Western blotting detection reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The RT PreMix kit was provided by Enzynomics (Daejeon, Korea). DSS was purchased from MP Biomedicals (Irvine, CA, USA).
10
Lung Protein Expression Analysis
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The lysates (30 μg/lane) of lung and cell culture protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (Millipore). The blots were incubated overnight with primary antibodies anti-α-ENaC, anti-TRAF6 (1:200, Santa Cruz Biotechnology, USA), anti-phosphorylated-p38, anti-p38, anti-phosphorylated-Erk, anti-Erk, anti-phosphorylated-JNK, anti-JNK, anti-phosphorylated-NF-κB p65, anti-IκB-α, anti-TATA (1:1,000, Cell Signaling Technology, USA), and anti-β-actin (1:10,000, Sigma Chemical Company, USA). The anti-phosphorylated NKCC1 and anti-NKCC1 antibodies were custom made by GeneTex.
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