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4 protocols using pan p38

1

Comprehensive Cell Signaling Analysis

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We used the following reagents in this study: antibiotics (mixture of 100 μg/mL of streptomycin and 100 units/mL of penicillin), Trizol, Ca2+- and Mg2+-free Hank’s balanced salt solution (GIBCO BRL, Gaithersburg, MD, USA); Eagle’s minimum essential medium (EMEM), McCoy’s 5a medium, and fetal bovine serum (FBS) (American Type Culture Collection (ATCC), Manassas, VA, USA); Bay 11-7085, SB203580, PD98059, and SP600125 (Calbiochem, La Jolla, CA, USA); GM6001 and Ponceau S (Sigma-Aldrich, St. Louis, MO, USA); and SR11302 (MedChemExpress, Monmouth Junction, NJ, USA).
The following antibodies were used in this study: rabbit monoclonal antibodies (mAbs) against phospho-IκBα and rabbit polyclonal Abs against phospho-p65, phospho-c-jun, pan-extracellular signal-regulated kinase 1/2 (ERK1/2, p44/p42), phospho-ERK1/2, pan-p38, phospho-p38, pan-JNK (p54/p46), and phospho-JNK (Cell Signaling Technology, Inc., Beverly, MA, USA); mouse mAb against human MMP-7 (R&D Systems, Minneapolis, MN, USA); rabbit polyclonal Ab against human syndecan-2 (Thermo Fisher Scientific, Waltham, MA, USA); and mouse mAbs against actin and lamin B, goat anti-mouse, and anti-rabbit secondary Abs conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
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2

Quantitative Western Blot Analysis

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Western blot analysis was performed according to established protocols [18] (link), [22] (link). Briefly, proteins extracted from heart tissue lysis were loaded to 10–15% SDS-PAGE, and then transferred to a nitrocellulose membrane (Bio-Rad). The blocking solution was composed of 3% BSA dissolved in 1×TBS; blocking the membrane for 1 h at room temperature. The membrane was incubated for 2 h at room temperature in primary antibody and 1 h in secondary antibody, both in 1.5% BSA dissolved in 1×TBS. The signals were detected with the ECL system (Amersham Biosciences), and the signals were quantified by scanning densitometry and computer-assisted image analysis. Pan p38, phospho-p38, pan ERK, phospho-ERK, pan JNK, phospho-JNK, pan STAT3, and phospho-STAT3 antibodies were from Cell Signaling Technology (Beverly, MA). Pan gp130, phospho-gp130, and GAPDH antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
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Molecular Signaling Pathway Analysis

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Antibiotics, phosphate-buffered saline (PBS) and TRIzol were achieved from GIBCO BRL (Gaithersburg, MD, USA). N-[4-[2,3-Dihydro-1-(2-methylbenzoyl)-1H-indol-5-yl]-5-methyl-2-thiazolyl]-1,3-benzodioxole-5-acetamide (ML385), bovine serum albumin (BSA), skim milk, Tween-20 and L-sulforaphane were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Cell Signaling Technology, Inc. (Beverly, MA, USA) supported Abs against several molecules such as phospho-c-jun (Cat No. 9164), phospho-p65 (Cat No. 3033), phospho-IκBα (Cat No. 2859), phospho-p38 (Cat No. 9211), phospho-ERK1/2 (Cat No. 9101), phospho-JNK (Cat No. 9251), pan-p38 (Cat No. 8690), pan-ERK1/2 (p44/p42) (Cat No. 9102), pan-JNK (p54/p46) (Cat No. 9252). Santa Cruz Biotechnology (Santa Cruz, CA, USA) supported mouse primary Abs against actin (sc-47778) and lamin B (sc-374,015) and goat anti-mouse (sc-2005) and anti-rabbit (sc-2004) Abs conjugated to horseradish peroxidase. Bioss Antibodies, Inc. (Woburn, MA, USA) supported rabbit anti-phospho-Nrf2 (bs-2013R). Chemical inhibitors such as PD98059, SB203580, SP600125 and Bay 11-7085 were purchased from Calbiochem (La Jolla, CA, USA). J14 and SR11302 were purchased from MedChemExpress (Monmouth Junction, NJ, USA) and Tocris Bioscience (Bristol, UK), respectively.
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4

Western Blot Analysis of Cardiac Proteins

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Western blot analysis was performed according to established protocols [18 (link), 22 (link)]. Briefly, proteins extracted from heart tissue lysis were loaded to 10–15% SDS-PAGE, and then transferred to a nitrocellulose membrane (Bio-Rad). The blocking solution was composed of 3% BSA dissolved in 1×TBS; blocking the membrane for 1 hour at room temperature. The membrane was incubated for 2 hours at room temperature in primary antibody and 1hour in secondary antibody, both in 1.5% BSA dissolved in 1×TBS. The signals were detected with the ECL system (Amersham Biosciences), and the signals were quantified by scanning densitometry and computer-assisted image analysis. Pan p38, phospho-p38, pan ERK, phospho-ERK, pan JNK, phospho-JNK, pan STAT3, and phospho-STAT3 antibodies were from Cell Signaling Technology (Beverly, MA). Pan gp130, phospho-gp130, and GAPDH antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
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