Recombinant human TGF-β protein (active; cat. no. ab50036) and SB203580 (P38 inhibitor; cat. no. ab120162) were obtained from Abcam. The mimic (cat. no. miR10000728-1-5) and inhibitor (cat. no. miR20000728-1-5) of miR-375 and their negative controls [mimic control (MControl, cat. no. miR1N0000001-1-5) and inhibitor control (IControl, cat. no. miR2N0000001-1-5)] were synthesized by Guangzhou RiboBio Co., Ltd. The small interfering RNA (siRNA) against Map2k6 (siMapk2k6; cat. no. sc-35913) was purchased from Santa Cruz Biotechnology, Inc. and scramble control RNA (siRNA, cat. no. sc-37007) was used as the control. Primary antibodies against the following proteins were obtained from Abcam: α-SMA (cat. no. ab32575, 1:1,000 dilution), periostin (cat. no. ab14041, 1:1,000 dilution), β-actin (cat. no. ab8226, 1:1,000 dilution), phosphorylated (p)-Smad3 (cat. no. ab52903, 1:1,000 dilution, total (t)-Smad3 (cat. no. ab40854, 1:1,000 dilution) and MAP2K6 (cat. no. ab33866, 1:1,000 dilution). Anti-p-AKT (cat. no. 4060, 1:1,000 dilution), anti-t-AKT (cat. no. 4691, 1:1,000 dilution), anti-p-P38 (cat. no. 4511, 1:1,000 dilution) and anti-t-P38 (cat. no. 9212, 1:1,000 dilution) were purchased from Cell Signaling Technology, Inc.
Anti t p38
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-t-P38 is a primary antibody that recognizes the total form of p38 MAPK, a serine/threonine protein kinase that plays a crucial role in various cellular processes such as stress response, inflammation, and cell differentiation. This antibody can be used to detect the expression levels of total p38 MAPK in biological samples.
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Lab products found in correlation
Anti p p38, by Cell Signaling Technology (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
α sma, by Abcam (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti cyto c, by Cell Signaling Technology (1 mentions)
Anti t erk1 2, by Cell Signaling Technology (1 mentions)
Anti t jnk, by Cell Signaling Technology (1 mentions)
Anti p jnk, by Cell Signaling Technology (1 mentions)
Anti nlrx1, by Proteintech (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
Ab124964, by Abcam (1 mentions)
Ab45688, by Abcam (1 mentions)
Ab124805, by Abcam (1 mentions)
Ab109520, by Abcam (1 mentions)
Anti col1, by Cell Signaling Technology (1 mentions)
Anti cd68, by Proteintech (1 mentions)
Anti mdm2, by Cell Signaling Technology (1 mentions)
Ecl kit, by 4A Biotech (1 mentions)
6 protocols using anti t p38
1
Transforming Growth Factor-β Pathway Regulation
2
Western Blot Analysis of Lung Proteins
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Lung tissues were gently homogenized and lysed in RIPA lysis and extraction buffer (Thermo Fisher). The protein concentrations were measured using the Bradford assay. Equal amounts of protein samples were loaded onto 8–12% SDS-PAGE gels and separated by electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). Membranes were blocked in Tris buffered saline containing 0.1% Tween 20 (TBST) with 5% skim milk and incubated overnight at 4 °C with primary antibodies. After washing with TBST, the membranes were incubated at room temperature with secondary antibodies for 1 h. The protein signal was analyzed using Image J software. Protein samples were normalized to GAPDH. The primary antibodies were used as follows: anti-NLRX1 (Proteintech, Rosemont, IL), anti-BAX, anti-Cyto C, anti-P-ERK 1/2, anti-T-ERK 1/2, anti-P-JNK, anti-T-JNK, anti-P-p38, anti-T-p38, and anti-GAPDH (Cell Signaling Technology, Danvers, MA).
3
Western Blot Analysis of Fibrosis Markers
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We performed western blot (WB) analysis as previously described36 using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd) and the following antibodies: anti‐GAPDH (1:5000, 60004‐1‐Ig; Proteintech, USA), anti‐αSMA (1:1000, ab124964; Abcam, UK), anti‐fibronectin (1:1000, ab45688; Abcam, UK), anti‐MDM2 (1:1000, #86934; Cell Signaling Technology, USA), anti‐t‐p53 (1:1000, 10442‐1‐AP; Proteintech, USA), anti‐Col1 (1:1000, #84336; Cell Signaling Technology, USA), anti‐vimentin (1:1000, #5741; Cell Signaling Technology, USA), anti‐CD68 (1:5000, 25747‐1‐AP; Proteintech, USA), anti‐p‐p38 (1:1000, #4511; Cell Signaling Technology, USA), anti‐t‐p38 (1:1000, #8690; Cell Signaling Technology, USA), anti‐p21 (1:1000, ab109520; Abcam, UK), and nti‐FSP1 (1:1000, ab124805; Abcam, UK).
4
Protein Extraction and Western Blot Analysis of Aortic Tissue
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Protein extraction from aortic tissue was done as previously described (Rivera-Torres, 2015 (link)). The cytosol and nuclear protein were obtained using Nuclear and Cytoplasmic Protein Extraction kit (CWBIO) according to the manufacturer’s instructions. Proteins were separated by SDS-PAGE and transferred electrophoretically to polyvinylidene fluoride membranes (Millipore). After blocking with 5% nonfat milk in TBST (pH 7.4, 20 mM Tris-HCl, 15 m NaCl, and 0.1% [vol/vol] Tween-20), the membranes were incubated overnight at 4°C with primary antibodies as follows: anti-TSH (bs-2676R) from Bioss; anti–NF-κB p65 (ab32536, clone E379), anti-IκBα (ab32518, E130) from Abcam; anti–p-IκBα (CST9246, clone 5A5), anti–p-Thr202/Tyr204 Erk1,2 (CST4695, clone 137F5), anti–t-ERK (CST4376, clone 20G11), anti–p-Thr180/Tyr182 p38MAPK (CST4511, clone D3F9), anti–t-p38 (CST9228, clone L53F8), anti–p-Thr183/Tyr185 JNK (CST4668 clone 81E11) from Cell Signaling Technology; anti–t-JNK (sc-7345, clone D-2) from Santa Cruz Biotechnology; and anti-GAPDH, (60004-1-ig, clone 1E6D9) and anti–laminin B (66095-1-ig, clone 3C10G12) from Proteintech. After washing with TBST, the membranes were incubated for 1 h at 25°C with the appropriate HRP-conjugated secondary antibody. The bands were visualized by FluorChemQ system using a chemiluminescence kit (Pierce).
5
Cytoplasmic and Mitochondrial Fractionation
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Cytoplasmic and mitochondrial fractional extracts were isolated using Mitochondria/Cytosol Fractionation Kit (BioVision). Total cell lysates or fractional extracts were electrophoresed, transblotted, and immunoblotted following standard procedures. Primary antibodies used include anti-FasL (Santa Cruz sc6237, 1:2000), anti-CCN1 (1:5000) (a generous gift from Shr-Jeng Leu, PhD; National Yang Ming University, Taiwan), anti-XIAP (R&D AF8221, 1:4000), anti-Caspase-3 (Cell Signaling 9662, 1:4000), anti-Caspase-8 (Cell Signaling 9429, 1:1000), anti-HtrA2 (R&D AF1458, 1:4000), anti-Smac (R&D AF7891, 1:1000), anti-pp38 (Cell Signaling 9211, 1:4000), anti-tp38 (Cell Signaling 9212, 1:4000), anti-COX IV (Cell Signaling 4844, 1:1000), and anti-β-tubulin (Abcam ab6046, 1:8000). General reagents and chemicals were from Sigma.
6
Apoptosis Pathway Protein Analysis
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To determine levels of proteins in the apoptosis pathway, we treated HEI-OC1 cells with 60 μM CDDP and 1 or 10 μg/ml ATX/ATX-LPN as described above, and then harvested the cells for western blot analyses. The protein extracts were subjected to 10% SDS-PAGE and electrotransferred to PVDF membranes. The membranes were then blocked for 1 h in quick-blocker at room temperature and incubated overnight in a cold chamber (4 °C) with specific primary antibodies. After incubation, the membranes were washed with TBS and then incubated with HRP conjugated secondary antibody for 1 h at room temperature, washed repeatedly, and visualized with an enhanced chemiluminescence kit (Thermo Fisher Scientific). GAPDH and α-tubulin served as internal standards. The relative intensity was quantified by Quantity One. The following primary antibodies were used: anti-cleaved-caspase 3, anti-cleaved-caspase 9, anti-p53, anti-BCL-2, anti-P-P38, anti-T-P38 and anti-cytochrome 3 (Cell Signaling Technology, CST), anti-P-JNK (Abcam), and anti-T-JNK (Abcam).
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