T p70s6 kinase

ASMCs were washed once with cold DPBS (#14190, Thermo Fisher Scientific) and lysed by RIPA buffer (#R0278, Sigma-Aldrich). Cell lysates were centrifuged at 12,000 rpm (10 min) and the protein concentration of the supernatant was measured by using the BCA protein assay kit (#23227, Thermo Fisher Scientific). Equal amounts of denatured proteins (20 μg) were separated in 4%–12% SDS–PAGE (#M41212, GenScript, Piscataway, NJ, USA), and, subsequently, transferred onto a nitrocellulose membrane (#88018, Themo Fisher Scientific). Proteins of interest were detected by specific antibodies, including total- (t-)PI3K (#4292), and phosphorylated- (p-)PI3K (#GTX132597), t-AKT (#4691) and p-AKT (#4060), t-p70S6 kinase (#2708) and p-p70S6 kinase (#9205), t-STAT3 (#9139) and p-STAT3 (#9145), PTEN (#9188, which were all purchased from Cell Signaling Technology, Cambridge, UK), PGC-1α (#ab54481, Abcam), t-PPARγ (#sc-7273, Santa Cruz), and p-PPARγ (#sc-28001, Santa Cruz). GAPDH (#2118, Cell Signaling Technology) was used as an endogenous control for a semi-quantification measure. Protein bands were visualized by Luminata Forte Western HRP substrate (#WBLUF, Sigma-Aldrich) after binding specie-specific secondary HRP conjugated antibodies (#7076 and #7074, Cell Signaling Technology).

Ang II was purchased from ENZO (ALX-151-039-M025); collagenase and trypsin were purchased from Gibco (Grand Island, NY, United States); the BCA protein assay kit was bought from Pierce (Rockford, United States); and 2,7-dichlorofluorescindiacetate (DCFH-DA) was obtained from the Bioengineering Institute (Nanjing, China). The following primary antibodies were obtained from Cell Signaling Technology (CST, United States): glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#2118), p-mTORC1 (#2971), T-mTORC1 (#2983), α-actinin (#69758S), P-p70 S6 kinase (Thr389) (#9234P), T-p70 S6 kinase (#2708), P-JNK (T183/Y185) (#4668P),T-JNK (#9258), P-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370P), T-ERK (#4695), P-p38 (#4511P), p38 MAPK (#9212P), T-TAK1 (#5206), P-TAK1 (#4508), T-AKT (#4691), P-AKT (#4060), acetyl-CoA carboxylase antibody (#3676), and P-acetyl-CoA carboxylase antibody (#3661S). ABCAM provided the following primary antibodies: Anti-AMPKα2 (ab3760), p-AMPKα2 (S491, ab109402), anti-SOD1 (ab16831), anti-SOD2 (ab38155), Nrf2 (ab15323), 4-hydroxynonenal (ab46545), sarcomeric α-actinin (ab68167), heme oxygenase1 (ab-13243), and NOX2/gp91phox (ab129068). The Sesn2 antibody was acquired from Proteintech (no. 10795-1-AP). Antibodies were used at 1:1,000 dilutions for Western blotting. The secondary antibodies were obtained from LI-COR Biosciences (Lincoln, United States).