Western blotting was performed as previously described.21 (link) The following primary antibodies were used: APOC1 (1:1000, ab198288), cyclin B1 (1:5000, ab32053), cyclin D1 (1:5000, ab134175), caspase-9 (1:1000, ab202068), BCL-2 (1:500, ab32124), ERK1/2 (1:500, ab196883), phospho-ERK1/2 (1:500, ab65142), and GADPH (1:2500, ab9485) were purchased from Abcam Inc. Phospho-p38 (1:1000, #9211), p38 (1:1000, #9212), phospho-JNK (1:1000, #9251) and JNK (1:1000, #9252) were purchased from Cell Signaling Technology (Boston, MA, USA). Goat anti-rabbit IgG H&L (1:2000, ab6721, Abcam) was used as a second antibody.
Phospho p38
Manufactured by Abcam
Sourced in United States, United Kingdom
Phospho-p38 is a lab equipment product that detects the phosphorylated form of the p38 MAPK protein. It is used to measure the activation of the p38 MAPK signaling pathway in various cellular and biochemical assays.
Automatically generated - may contain errors
Lab products found in correlation
Phospho jnk, by Abcam (9 mentions)
β actin, by Abcam (6 mentions)
Gapdh, by Abcam (5 mentions)
Phospho erk1 2, by Abcam (3 mentions)
Phospho erk, by Abcam (3 mentions)
Caspase 3, by Abcam (3 mentions)
Phospho p65, by Abcam (2 mentions)
Phospho erk1 2, by Santa Cruz Biotechnology (2 mentions)
Phospho ikbα, by Abcam (2 mentions)
Erk1 2, by Santa Cruz Biotechnology (2 mentions)
Bcl 2, by Abcam (2 mentions)
Bcl2 associated x (bax), by Abcam (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
C fos, by Abcam (2 mentions)
Phospho iκbα, by Abcam (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ab198288, by Abcam (1 mentions)
Ab32053, by Abcam (1 mentions)
Ab134175, by Abcam (1 mentions)
Ab202068, by Abcam (1 mentions)
18 protocols using phospho p38
1
Comprehensive Western Blotting Protocol
2
Protein Expression Analysis by Western Blot
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The lysed cell homogenates were centrifuged at 13,000 ×g for 10 min at 4°C to remove cell debris. Total proteins were collected and the protein concentrations were determined using the Bradford method. Proteins (20 μg) were separated on 10% SDS-PAGE and transferred to polyvinyl difluoride membranes (Millipore, Bedford, MA). Membranes were blocked with 5% milk in TBS-T (TBS containing Tween 20) for 30 min and incubated with primary antibodies overnight at 4°C. Primary antibodies were all obtained from Abcam (Cambridge, MA) and used at the following dilutions: hTERT (1 : 1000), phospho-JNK1 and phospho-JNK2 (pT183 and pY185, resp.; 1 : 1000), NK1/2 (1 : 500), phospho-c-Jun (pS63; 1 : 5000), c-Jun (1 : 1000), phospho-p38 (pT180; 1 : 1000), p38 (1 : 200), pro-caspase-9 (1 : 2000), cleaved caspase-9 (1 : 2000), pro-caspase-3 (1 : 1000), cleaved caspase-3 (1 : 500), GAPDH (1 : 10000), and β-actin (1 : 2500). After washing three times with TBS-T for 10 min, membranes were incubated with goat polyclonal anti-rabbit IgG-H&L-preadsorbed (HRP) secondary antibody at room temperature for 1 h. Protein bands were visualized by chemiluminescent detection and the densitometric values were determined using a gel image analysis system (Bio-Rad, Hercules, CA). Data were normalized to GAPDH or β-actin.
3
Protein Expression Analysis by Western Blot
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Total cell and tissues lysates were prepared in 1× sodium dodecyl sulfate buffer. Identical quantities of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose filter membranes. The blots were incubated with antibodies specific for CBX6 (Abcam, CA, USA), S100A9 (Abcam, CA, USA), total-ERK1/2(Abcam, CA, USA), phospho-ERK1/2 (Abcam, CA, USA),total-p38(Abcam, CA, USA), phospho-p38(Abcam, CA, USA), total-p65 (Abcam, CA, USA), phospho-p65 (Abcam, CA, USA), total-p50 (Abcam, CA, USA), phospho-p50 (Abcam, CA, USA), or GAPDH (Abcam, CA, USA), after which they were incubated with IRDye 800-conjugated goat anti-rabbit IgG and IRDye 700-conjugated goat anti-mouse IgG, and the signals were detected using an Odyssey infrared scanner (Li-Cor). GAPDH was used as a loading control for these experiments.
4
Western Blot Analysis of Protein Expression in BMDCs
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The expression of proteins of interest in BMDCs was analyzed using western blotting as previously described 1 (link). The following primary antibodies were used: anti-TGR5 (Abcam, Cambridge, MA, USA), FXR (Abcam), Bax (Abcam), BCL-2 (Abcam), caspase3 (Abcam), cleaved-caspase3 (Abcam), LC3 (Abcam), Belin1(Abcam), P62 (Abcam), P65 (Abcam), Ikbα (Abcam), ERK1/2 (Santa Cruz), P38 (Abcam), JNK (Abcam), phospho-P65 (CST), phospho-Ikbα (Abcam), phospho-ERK1/2 (Santa Cruz), phospho-P38 (Abcam), phospho-JNK (Abcam), and β-actin (Abcam).
5
Quantifying TGF-β1 and SMAD Signaling
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Tissue protein extracts were generated using RIPA buffer (150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 10 mM NaF, 50 mM Tris pH 7.4) with protease (cOmplete tablets, EDTA free, Roche) and phosphatase (phosphatase inhibitor cocktail sets I + II, Millipore) inhibitors. Standard Western blotting analysis was performed using 10% SDS-PAGE gels with the following primary antibodies from Cell Signaling: SMAD3 (1:1000, #9523), Phospho-SMAD3 (1:1000, #9520), SMAD2 (1:1000, #5339), Phospho-SMAD2 (1:1000, #3108), ERK1/2 (1:1000, #4695), Phospho-ERK1/2 (1:2000, #4370), p38 (1:1000, #8690), and Phospho-p38 (1:1000, #4511), and the following antibody from Abcam: Total OXPHOS Rodent WB Antibody Cocktail (1:5000, ab110413). Secondary antibody incubations were performed at room temperature for 60 min using HRP-conjugated antibodies (1:10,000) and then imaged using a ChemiDoc system (Bio-Rad).
Quantification of the TGF-β1 content was performed on cardiac tissue and plasma using an ELISA kit by R&D Systems (DB100B) according to the manufacturer's instructions. The total TGF-β1 amounts were measured by first activating the samples (to convert biologically inactive latent TGF-β1 into active TGF-β1) as instructed by the kit protocol, while measurement of the “bioavailable” TGF-β1 content was performed by skipping the initial activation step.
Quantification of the TGF-β1 content was performed on cardiac tissue and plasma using an ELISA kit by R&D Systems (DB100B) according to the manufacturer's instructions. The total TGF-β1 amounts were measured by first activating the samples (to convert biologically inactive latent TGF-β1 into active TGF-β1) as instructed by the kit protocol, while measurement of the “bioavailable” TGF-β1 content was performed by skipping the initial activation step.
6
Protein Extraction and Western Blot Analysis
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The total proteins of tissues and cell samples were extracted using a radioimmunoprecipitation assay buffer (Solarbio. Equal amounts of protein (20 μg) were separated by sodium dodecyl sulfate–, Beijing, China)polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking with 5% bovine serum in TBST, the membranes and corresponding primary antibodies were incubated overnight in a shaker at 4°C. The primary antibodies used were as follows: anti-GAPDH (1:10,000, ProteinTech, Wuhan, China), anti-HSF2BP (1:1000, Abcam, USA), ERK (1:500, Abcam, USA), phospho-ERK (1:500, Abcam, USA), JNK (1:1,000, Abcam, USA), phospho-JNK (1:1,000, Abcam, USA), p38 (1:1,000, Abcam, USA), and phospho-p38 (1:1,000, Abcam, USA). After incubation with horseradish peroxidase-conjugated secondary antibodies (1:10,000, ProteinTech, Wuhan, China) at room temperature for 1 h, the membranes were visualized using the enhanced chemiluminescence method (Thermo, MA, USA) with a chemiluminescent detection system (Tanon, Shanghai, China).
7
Western Blot Analysis of Lung Tissue
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Right lung (40 mg) was cut off. Then, 400 μL of CER I and 4 μL of Halt™ Protease and Phosphatase Inhibitor Cocktail (Pierce Biotechnology Inc., Rockford, USA) homogenate were added to the lung tissue. The mixture was incubated on ice for 10 min followed by addition of precooled (4°C) CER II (22 μL). The supernatant was collected by centrifugation for 10 min at 16000 rpm. Protein centration was measured by BCA method. Equal amounts of proteins were loaded in a 12% SDS-PAGE gel. After blotting onto PVDF membrane, samples were incubated with respective primary antibodies: phospho-ERK, C-jun (Cell Signaling Technology, Boston, MA), phospho-p38, phospho-JNK, ERK, p38, JNK, C-fos, γ-GCS-h (Abcam, Cambridge, UK), and β-actin (1 : 1000 dilution) and were incubated at 4°C overnight. Then, the respective secondary antibodies conjugated to HRP were incubated for 1 h, followed by three-time washing. The membrane was then incubated with ECL (Millipore, MA, USA) for luminescence generation. The image and the gray level were analyzed by Alpha Innotech and Adobe software. Protein expression level was evaluated by the β-actin ratio.
8
Protein Isolation and Western Blot Analysis
9
Western Blot Analysis of Protein Expression
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Western blotting was performed essentially as previously described (17 (link)). The expression of CD137 (1:500, rat, Abcam, Cambridge, UK), ASK1 (1:2,000, rabbit, Abcam), phospho-ASK1 [1:500, rabbit, Cell Signaling Technology (CST), Danvers, MA, USA], p38 (1:1,000, rabbit, CST), phospho-p38 (1:1,000, rabbit, CST), JNK (1:1,000, rabbit, CST), phospho-JNK (1:1,000, rabbit, CST), IκBα (1:1,000, rabbit, CST), phospho-IκBα (1:1,000, rabbit, CST), MMP9 (1:1,000, rabbit, Abcam), MMP12 (1:2,000, rabbit, Abcam), and β-actin (1:1,000, rabbit, CST) in MH-S cells or lung tissues was measured by western blot. Protein expression was normalized to β-actin.
10
Protein Extraction and Western Blot Analysis from Mouse Nervous Tissues
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Extraction of total tissue protein: all operations were performed on ice. Mouse DRG tissue and sciatic nerve tissue were removed from the -80°C ultralow temperature refrigerator, and 500 μL RIPA strong lysate and 5 μL protease were added to the tissue of each mouse at a ratio of 100 : 1 inhibitor (PMSF) mixture. After grinding the homogenate, centrifugation was performed in a low temperature centrifuge at 12000 rpm and 4°C for 15 min, the supernatant was slowly removed, and the protein concentration was measured using a BCA kit (P0010; Beyotime, China) on a microplate reader (Infinite M200 Pro; Tecan, Switzerland). The proteins were analyzed by 8-12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). Nonspecific binding sites were blocked with 5% (w/v) BSA (4240GR100; BioFroxx), and the membranes were incubated with the primary antibodies NF-κB, ERK1/2 (Abcam), phospho-p38, phospho-SAPK/JNK, phospho-ERK1/2, p38, SAPK/JNK, and GAPDH (Cell Signaling Tech). The membranes were washed 4 times in TBST and then incubated with the secondary antibodies for 1 h at room temperature on a shaker. Protein expression was measured by using an ECL system (UV200R; Tanon).
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