Anti total p38 mapk

Western immunoblots were run as described previously [24 (link)]. Primary antibodies and their sources were as follows. Anti-total p38 MAPK, anti-phosphorylated p38 (pp38) MAPK, anti-Erk, anti-phosphorylated Erk, anti-activated caspase 3, anti-activated caspase 8 were from Cell Signaling Technology. Anti-β-actin and the secondary antibodies horseradish peroxidase-conjugated anti-rabbit IgG and anti-rabbit IgG were from Sigma-Aldrich. Anti-goat IgG was from Santa Cruz Biotechnology.

Protein was extracted from cleaned and snap frozen mesenteric arteries and aortas from WKY, SHRSP rats, and from cultured rat VSMCs. Protein (30 μg) was separated by electrophoresis on a polyacrylamide gel and transferred to a nitrocellulose membrane. Non-specific binding sites were blocked with 5% bovine serum albumin in Tris-buffered saline (TBS) solution. Membranes were then incubated with specific antibodies overnight at 4 °C. Membranes were washed 3 times with TBS-Tween 20 and incubated with specific secondary antibodies for 1 h at room temperature. Signals were revealed after reaction with enhanced chemiluminescence. Results were normalised to α-tubulin or β-actin, as indicated in the figures and are expressed as arbitrary units. In most of our studies we used α-tubulin as the housekeeping protein, except for the studies assessing p66Shc expression in aorta, where we used β-actin as our internal control. This related to technical aspects, where β-actin detection was superior to that of α-tubulin. Antibodies were as follows: anti-TGFβ (SC146, Santa Cruz, USA) anti-fibronectin (F3648, Sigma-Aldrich, UK), anti-phospho-p66Shc (566,807, Calbiochem, USA), anti-α-tubulin (AB4074, Abcam, UK), anti-phospho-p38MAPK (9211S, Cell Signaling, UK) anti-total-p38MAPK (9212S, Cell Signaling, UK), Nox1 (sc-25,545, Santa Cruz, USA) and anti-β-actin (ab8229, AbCam, UK).

Immediately after ex vivo analysis, RV tissue was then washed in ice‐cold saline and snap‐frozen. RV tissue was then homogenized using an Omni international tissue grinder (Thermo Fisher Scientific, Waltham, MA) in ice‐cold RIPA lysis buffer (Thermo Fisher) containing proteinase inhibitor cocktail (EMD Millipore/Sigma‐Aldrich, St. Louis, MO) and PhosStop inhibitor cocktail (Roche, Indianapolis, IN). After homogenization, lysate was sonicated for ten one‐second pulses at 100% power and then centrifuged. The supernatant was saved and used as whole lung lysate. Protein concentration was measured using BCA Protein Assay (Thermo Fisher). Rabbit polyclonal anti‐phospho‐p38MAPK, anti‐total p38MAPK, anti‐bcl2, and anti‐bax (all used at 1:1000, and from Cell Signaling, Danvers, MA) and mouse monoclonal anti‐Vinculin loading control (1:5000; Calbiochem; Billerica, MA) primary antibodies were used, all diluted in Pierce Protein‐Free T20 Blocking Buffer (Thermo Fisher). All antibodies used have been extensively validated (Lin et al. 2005; Bernal‐Mizrachi et al. 2006; Bikkavilli et al. 2008; Tang et al. 2008; Slone et al. 2011; Choi et al. 2016; Jiang et al. 2016; Zhang et al. 2017). Rabbit‐HRP (Cell Signaling) and mouse‐HRP (KPL, Gaithersburg, MD) secondary antibodies were diluted 1:2000 in Pierce Protein‐Free T20 Blocking Buffer (Thermo Fisher). Densitometry was performed using ImageJ.

Amaxa^® Cell Line Nucleofector^® Kit T and Amaxa^® Glia Cell Nucleofector^® Kit T were purchased from LONZA. Primers for quantitative RT-PCR were synthesized by Life Technologies. SYBR Green for quantitative RT-PCR was purchased from Roche. SP600125, Bay11–7082, SB203580, U0126 and LPS were purchased from Sigma. Amyloid-β 42 (Aβ42) peptide was purchased from AnaSpec. Oligomeric Aβ42 was prepared as previously described (Huang et al., 2015 (link)). Antibodies used in this study are as followed: anti-phospho-p38-MAPK, anti-total-p38-MAPK, anti-phospho-ERK1/2, anti-total-ERK1/2, anti-phospho-JNK, anti-total-JNK, anti-phospho-IκBα, anti-total-IκBα, anti-phosho-NF-κB, anti-total-NF-κB, anti-phospho-c-Jun, anti-total-c-Jun and anti-β-actin were purchased from Cell Signaling Technology; anti-tubulin (Millipore); anti-mouse IgG and anti-rabbit IgG antibody conjugated with horseradish peroxidase (ThermoFisher Scientific).