Journal: bioRxiv

Article Title: Targeting C42 in PKGIα alleviates diastolic dysfunction in HFpEF

doi: 10.1101/2025.05.09.653217

Figure Lengend Snippet: A , PKGIα disulfide dimerization and VASP Ser239 phosphorylation in vascular smooth muscle cells treated with quercetin, retinol, retinoic acid, urolithin A or ergothioneine in the presence or absence of Br-cGMP (n=6). B , PKGIα disulfide dimerization and VASP Ser239 phosphorylation in vascular smooth muscle cells treated with quercetin in the presence or absence of Br-cGMP (n=5). *P<0.05; **P<0.01; ***P<0.005. Comparisons were made using 1-way ANOVA followed by the Tukey post hoc test.

Article Snippet: Samples were immunoblotted for Phospho-VASP (Ser239) (Cell Signaling, #3114), Phospho-PLN (Ser16) (Badrilla, #A010-12AP), ANP (Proteintech, #27426-1-AP), a/b-Tubulin (Cell Signaling, #2148S) or GAPDH (Cell Signaling, #2118).

Techniques: Phospho-proteomics

Journal: Diabetes & vascular disease research

Article Title: Pancreatic-β-cell survival and proliferation are promoted by protein kinase G type Iα and downstream regulation of AKT/FOXO1.

doi: 10.1177/1479164117713947

Figure Lengend Snippet: Figure 7. Western blot analysis of RINm5F β-cells showing (a) 70% reduction in PKG-Iα protein levels caused by knockdown using two different siRNA constructs, PKG-Iα-siRNA 1 and 2, and dramatic reduction in PKG-Iα kinase activity, measured by VASP serine-239-phosphorylation, (b) quantification of PKG-I expression and VASP phosphorylation, (c) effects on key components of the AKT/FOXO1/PDX-1 pathway and (d) quantification of the westerns in (c). Phosphorylation/activation of eNOS was also included as a downstream phosphorylation target of AKT, indicating AKT kinase activity. Western blot analysis showing the effects of siRNA- mediated knockdown of PKG-Iα expression using siPKG-Iα S2 on components of the AKT/FOXO1/PDX-1 pathway in (e) INS-1 and (f) 1.1B4 β-cells.

Article Snippet: Apoptosis was measured by Cell Death Detection ELISAPLUS (Roche Applied Science) as described previously.38 Assessment of de novo DNA synthesis by BrdU ELISA DNA synthesis was measured by 5′-bromo-2′-deoxyuridine (BrdU) ELISA (Roche Applied Science) as described previously.32 Assessment of cell proliferation by MTT assay Proliferation was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Roche) as described previously.34 Caspase-3/7 activity by chemiluminescence assay Caspase-3/7 activity was determined using Caspase-3/7 Glo kit (Promega, Madison, WI, USA) as described previously.38 Protein extraction and western blotting Protein extraction/western blotting was performed as described previously.38 Blots were probed with primary antibodies: PKG-Iα/β (1:1000), p-(ser239)-VASP (1:500), total vasodilator-stimulated phosphoprotein (VASP) (1:2000) (or 1:1000 for total VASP from Santa Cruz Biotechnology), p-(ser1177)-eNOS (1:1000), total endothelial nitric oxide synthase (eNOS) (1:1000), PDX-1 (1:1000), p-(ser256)-Foxo1 (1:500), total Foxo1 (1:1000), p-(thr308)Akt (1:1000), p-(ser473)-Akt (1:1000), total Akt (1:1000), p-(ser133)-CREB (1:500), total cAMP response element binding protein (CREB) (1:1000), all from Cell Signaling Technology, β-actin (1:2000) (Santa Cruz Biotechnology), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000) (EMD Millipore) and subsequently with secondary antibodies labelled with infrared fluorescent dyes (LI-COR Biosciences, Lincoln, NE, USA) (1:25,000 in blocking buffer at room temperature for 1 h).

Techniques: Western Blot, Knockdown, Construct, Activity Assay, Phospho-proteomics, Expressing, Activation Assay