Yeast cells were grown to mid-log phase in YPD liquid medium. Flu was added to the medium, and 1 ml aliquots were withdrawn at the indicated time points. Cells were resuspended in sodium dodecyl sulphate (SDS) buffer (50 mM Tris-HCl [pH 6.8], 10% Glycerol, 2% SDS, 5 mM NaF, 1 mM Na3VO4 and 5% β-mercaptoethanol), boiled for 10 min and centrifuged at 13 000 x g for 10 min to obtain protein extracts. Protein concentration was measured using the RC DC Protein Assay kit (Bio-Rad), and 20 μg of total protein was loaded onto 10% Mini-PROTEAN TGX precast gels (Bio-Rad) and blotted on nitrocellulose membranes (Bio-Rad). Phosphorylated Hog1 was detected using anti-phospho-p38 MAPK antibody (Cell Signaling Technology) with 1:2000 dilution and IRDye 800CW donkey antibody against rabbit IgG (LI-COR Biosciences) with 1:5000 dilution. Total Hog1 protein was detected using anti-Hog1 yC-20 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) with 1:2000 dilution and IRDye 680RD donkey antibody against goat IgG (LI-COR Biosciences) with 1:5000 dilution. Signals were detected using the Odyssey Infrared Imaging System and quantified using the Odyssey 2.1 software (LI-COR Biosciences). All phosphorylated Hog1 values were normalized against the 5-min sample taken from the control strain.
Anti phospho p38 mapk antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-phospho-p38 MAPK antibody is a laboratory reagent used to detect the phosphorylated form of the p38 mitogen-activated protein kinase (MAPK) in various experimental techniques. This antibody specifically recognizes the phosphorylated form of p38 MAPK, which is a key signaling protein involved in cellular stress response pathways.
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Lab products found in correlation
Anti p38 mapk antibody, by Cell Signaling Technology (7 mentions)
Anti gapdh antibody, by Abcam (3 mentions)
Anti akt antibody, by Cell Signaling Technology (2 mentions)
Chemiluminescence reagent, by Cytiva (2 mentions)
Anti cd147 antibody, by Abcam (2 mentions)
Anti e cadherin antibody, by Abcam (2 mentions)
Anti fn antibody, by Abcam (2 mentions)
Anti cypa antibody, by Abcam (2 mentions)
Anti α sma antibody, by Abcam (2 mentions)
Goat anti rabbit igg, by Abcam (2 mentions)
Odyssey 2, by LI COR (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Rc dc protein assay kit, by Bio-Rad (1 mentions)
Bcip nbt, by Beyotime (1 mentions)
Alexa fluor 680 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti p38 mapk, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
12 protocols using anti phospho p38 mapk antibody
1
Quantifying Hog1 Phosphorylation in Yeast
2
Quantification of p38 MAPK Activation
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Intestinal tissue was isolated and homogenized in lysis buffer (Beyotime Biotechnology, China) on ice. The homogenate was centrifuged (14 000 rpm, 15 min, 4°C) and the level of total supernatant protein was measured by BCA protein assay kit (Beyotime Biotechnology, China). The protein samples (20 μg) were boiled in Laemmle’s sampling buffer and electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels. Next, the proteins were transferred to a polyvinylidene difluoride membrane and blocked with Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20. They were then incubated with anti-p38 MAPK or the anti-phospho-p38 MAPK antibody (1: 1000; Cell Signaling Technology, Danvers, MA) for 16 h at 4°C, followed by incubation with Alexa Fluor 680-conjugated anti-rabbit IgG (Molecular Probes). After washing 5 times for 5 min each time, the membranes were visualized by NBT/BCIP (Beyotime Institute of Biotechnology, Nantong, China). The protein bands were scanned and quantified using ImageJ software version 1.34 s (Wayne Rasband National Institute of Health) using β-actin for normalization.
3
Immunohistochemical Analysis of NSCLC Tissue
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Serial sections of a human lung tumor tissue array containing 116 NSCLC tumor tissue samples (60 squamous cell carcinoma and 56 adenocarcinoma specimens) and 16 normal/normal adjacent lung tissue samples (N/NAT) (4N and 12 NAT specimens) were purchased from Alenabio (LC121c and LC241h). H&E and immunohistochemical staining were performed according to previously described protocols,42 (link) using a 1:1000 dilution of an anti-WIP1 antibody (Abcam, ab31270), 1:100 dilution of an anti-phospho-p38 MAPK antibody (Cell Signaling Technology, #4511), 1:100 dilution of an anti-SOX2 antibody (Proteintech, 11064-1), and 1:100 dilution of an anti-ALDH1A1 antibody (Santa Cruz, sc-374149). The images were acquired with an optical microscope fitted with a camera (Olympus, Japan). To evaluate the expression of WIP1, p-p38, SOX2, and ALDH1 in normal or tumor tissue samples, staining scores were evaluated by multiplying the positive staining area (scored as 1: 0–25%; 2: 26–50%; 3: 51–75%; 4: 76–100%) with the staining intensity (scored as 1: weak; 2: moderate; 3: strong; 4: very strong).
4
Phosphorylation of Signaling Proteins
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A total of 10 µg protein was added to each well and was subjected to 10% SDS-PAGE under reducing conditions, and the separated proteins were transferred to polyvinylidene fluoride transfer membranes. Following blocking in TBS-Tween-20 (0.1%) buffer with 5% skim milk for 2 h at room temperature, the membranes were incubated at 4°C overnight with an anti-phospho-AKT (1;1,000; cat. no. 4051; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-phospho-p44/42 MAPK (1;1,000; cat. no. 4370; Cell Signaling Technology, Inc.), anti-phospho-SAPK/JNK (1;1,000; cat. no. 4668; Cell Signaling Technology, Inc.), or anti-phospho-p38 MAPK antibody (1;1,000; cat. no. 4511; Cell Signaling Technology, Inc.). Then the membranes were washed and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody (American Qualex, San Clemente, CA, USA). Following washing, the blots were visualized using SuperSignal West Dura Extended Duration substrate (Thermo Fisher Scientific, Inc.), and bands were detected using a myECL Imager system (version 2.0; Thermo Fisher Scientific, Inc.). Next, the same membranes were re-probed with anti-β-actin (Sigma-Aldrich; Merck KGaA), anti-AKT, anti-p44/42 MAPK (Erk1/2), anti-SAPK/JNK or anti-p38 MAPK antibody (Cell Signaling Technology, Inc.) to confirm equal loading of the proteins. All western blot analyses were performed in triplicate.
5
Western Blot Analysis of Signaling Pathways
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C7 cells treated under various conditions were lysed with lysis buffer (20 mM Tris/HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 100 mM NaF, 1% NP40, 1 μg/ml leupeptin, 1 μg/ml antipain and 1 mM PMSF). The protein content of this cell lysate was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA). An aliquot of each extract (40 μg of protein) was fractionated by electrophoresis in an SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membranes (Amersham, Arlington Heights, IL, USA). Membranes were blocked with a solution containing 3% skim milk, and then incubated overnight at 4°C with each of the following antibodies: anti-phospho-extracellular signal-regulated kinase (ERK) 1/2 antibody, anti-phospho-Akt antibody, anti-phospho-p38MAPK antibody, anti-ERK1/2 antibody, anti-Akt antibody, and anti-p38MAPK antibody (Cell Signaling Technology, Beverly, MA, USA). Subsequently, the membranes were incubated for 1 h at room temperature with anti-rabbit IgG sheep antibody or anti-mouse IgG sheep antibody coupled to horseradish peroxidase (Amersham). Reactive proteins were visualized using a chemiluminescence (ECL-plus) kit (Amersham) according to the manufacturer’s instructions.
6
Western Blot Analysis of Osteoblast Proteins
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Protein was extracted from cultured MC3T3-e1 cells using RIPA buffer. Cell lysates containing 20 µg of protein were mixed with sample buffer solution (Fujifilm) and separated on NuPAGE 4–12%® Bis–Tris Gels (ThermoFisher Scientific). Proteins were electrophoretically transferred onto iBlot 2 membranes (ThermoFisher Scientific) for dry protein transfer and then washed with DDW. The membranes were blocked with 4% Block Ace (KAC Co., Ltd., Japan) at RT for 1 h. The membranes were then incubated overnight at 4 °C with rabbit polyclonal anti-Hsp25 antibody (ab202846, Abcam, Tokyo, Japan), rabbit polyclonal anti-p38 MAPK antibody (#9212, Cell Signaling, MA, USA) 1:2000, rabbit polyclonal anti–phospho-p38 MAPK antibody (#9211, Cell Signaling) 1:800, rabbit monoclonal anti-Sp7/OSX antibody (ab209484, Abcam) 1:1,000, or mouse monoclonal anti–β-actin antibody (A3854, Sigma-Aldrich, Tokyo, Japan) 1:200,000. Membranes were then reacted with HRP-goat anti-rabbit IgG antibody (DK-2600, Dako, Glostrup, Denmark) 1:5000 for 1 h. Protein bands were visualized by chemiluminescence using an ImageQuant LAS4000 imaging system (GE Healthcare, Fairfield, CT, USA). Densitometric analysis was performed using ImageQuant LAS 4000 software. β-Actin was used as an internal standard in each lane for normalization of target protein expression.
7
Profiling Phosphorylated Signaling Proteins
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Cell lysates were extracted with a lysis buffer (20 mM Tris-HCl (pH = 8.0; FUJIFILM Wako), 150 mM NaCl (FUJIFILM Wako), 2 mM EDTA (FUJIFILM Wako), 100 mM NaF (FUJIFILM Wako), 1% NP40 (FUJIFILM Wako), 1 μg/mL leupeptin (Sigma), 1 μg/mL antipain (Sigma), and 1 mM phenylmethylsulfonyl fluoride (Sigma)). Phosphorylated protein and total protein in these lysates were examined by western blotting assay by using following primary antibodies: anti-phospho-Met (Tyr1234/1235) antibody, anti-phospho-Met antibody (Tyr1349), anti-Met antibody, anti-phospho-ERK1/2 antibody, anti-ERK1/2 antibody, anti-phospho-Akt antibody, anti-Akt antibody, anti-phospho-p38MAPK antibody, anti-p38MAPK antibody, anti-phospho-NF-κB antibody, anti-NF-κB antibody (Cell Signaling Technology, Beverly, MA, USA), anti-RANKL antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin antibody (Sigma). Proteins were visualized using Luminata Forte (Merck Millipore, Nottingham, UK) according to the manufacturer’s instructions.
8
Western Blot Analysis of Protein Markers
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Western blot analysis was performed as previously described [6 (link), 29 (link)]. The antibodies were diluted in 3 % blocking agent: anti-phospho-p38 MAPK Antibody (1:1000, Cell Signaling, Danvers, MA), Anti-PI3K p85 (phosphor Y607) antibody (1:1000, Abcam, Cambridge, UK), anti-phospho-PKA substrate antibody (1:10,000; Cell Signaling Technology, MA, USA), anti-phosphotyrosine antibody (PY20, 1:2,500, Abcam), anti- GAPDH antibody (1:1000, Abcam), anti-UQCRFS1 antibody (1:15,000, LSBio, Inc.), anti- PRDX5 antibody (1:2000, Abcam), anti-GPX4 antibody (1:15,000, Abcam), anti-ACTB antibody (1:500, Abcam), and anti-GSTM5 antibody (1:5,000, Abcam). Anti-α-tubulin mouse antibody (1:1000, Abcam) was used as the loading control for all western blots. The proteins on the membranes were detected with by an enhanced chemiluminescence (ECL) technique using ECL detection reagents.
9
Anti-inflammatory Effect of Compound
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The following reagents were used in this research: Mouse macrophage cell line RAW264.7 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China); Dulbecco's Modified Eagle's Medium (DMEM) and neutral red staining solution were bought from Solarbio (Beijing, China); Lipopolysaccharide (LPS) were obtained from Sigma-Aldrich (St. Louis, MO, USA); Griess kit and ROS kit were purchased from Beyotime Biotechnology (Shanghai, China); Mouse TNF-α and IL-6 ELISA kit were purchased from Beijing 4A Biotech Co, Ltd (Beijing, China); Primer iNOS, TNF-α, IL-6 and Cox-2 were designed and synthesized by Thermo Fisher Scientific (Shanghai, China Thermo Fisher scientific (Shanghai, China); Evo M-MLV RT Kit with gDNA Clean and TB Green TM Ex TaqTM II (Tli RNadeH Plus), Bulk kit were obtained from TaKaRa; anti-NF-κB p65 antibody, anti-phospho-NF-κB p65 antibody, anti-iNOS antibody, anti-COX-2 antibody, anti-IκBα antibody, anti-phospho-IκBα antibody, anti-P38 MAPK antibody, anti-phospho-P38 MAPK antibody, anti-Erk antibody, anti-phospho-Erk antibody, anti-JNK antibody and anti-phospho-JNK antibody were bought from Cell Signaling Technology (Danvers, MA, USA).
10
Murine Model of Cytokine Regulation
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OVA powder was purchased from Sigma-Aldrich (Grade V; St Louis, MO, USA). Adjuvant aluminum hydroxide (Al(OH)3(Alum)) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant human IL-27 antibody (rhIL-27), recombinant mouse IL27 antibody (rmIL-27), recombinant human IL-4 antibody (rhIL-4), recombinant mouse IL-4 antibody (rmIL-4), recombinant human IL-2 antibody (rhIL-2), recombinant mouse IL-2 antibody, anti-human INF-γ antibody (rmIL-2), anti-mouse INF-γ antibody, anti-human IL-4 antibody and anti-mouse IL-4 antibody were obtained from R&D Systems (Minneapolis, MN, USA). Anti-human CD3 antibody, anti-mouse CD3 antibody, anti-human CD28 antibody and anti-mouse CD28 antibody were obtained from eBioscience (San Diego, CA, USA). The following antibodies were also used: anti-STAT1 antibody (SC592; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Py-STAT1 antibody (catalog no. 9171; Cell Signaling, Boston, MA, USA), anti-GADD45γ antibody (ab196774,Abcam,Cambridge,UK), anti-p38 MAPK antibody (CST9212, Cell Signaling, Boston, MA, USA), and anti-phospho-p38 MAPK antibody (CST9215, Cell Signaling, Boston, MA, USA). The concentrations of IL-4, IL-5, and IL-13 were determined using ELISA kits (eBioscience).
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