Anti tgfβ sc146

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).

Fresh tissues were homogenized and centrifuged and soluble supernatants were taken as whole tissue lysates. Standard Western blots were performed using β-actin as the loading control. Antibodies used were anti-TGF-β (sc-146, Santa Cruz Biotechnology), anti-TNF-α (107,590–1-AP, Proteintech), anti-p65NF-κB (10,745–1-AP, Proteintech), anti-P-p65-S276 (AP0123, Abclonal) and anti-P-p65-S536 (AP0475, Abclonal), all used according to manufacturer recommended dilutions. For Western blot, 10–15 µg of whole lung tissue lysate per sample was used. The relative abundance of a variety of 40 cytokines were examined using mouse cytokine proteome profiler array (ARY028, R&D Systems). The whole tissue lysates of 4 mice in each group were used. Equal amount of total protein 200 µg per sample was hybridized to the array and compared for relative expression. The relative expression was quantified by measuring the dot blot intensity using Image J software.