Journal: bioRxiv

Article Title: Deletion of RSK2 kinase alleviates age-dependent hypertension

doi: 10.1101/2025.03.12.642932

Figure Lengend Snippet: (A) Representative western blot and bar graph showing RSK2 protein expression in mesenteric arteries from young (Y) RSK2 +/+ , aged (A) RSK2 +/+ , and A RSK2 -/- mice. Liver homogenate = a positive control, and GAPDH a loading control. A significant reduction in RSK2 expression was detected in A RSK2 -/- mice but not between Y and A RSK2 +/+ mice (n = 10 per group). (B) SBP was significantly lower in A RSK2 -/- mice compared to A RSK2 +/+ mice at baseline and following L-NAME treatment (n = 7-9 mice per group). (C) Heart rates were NS between A RSK2 -/- and A RSK2 +/+ mice at baseline or following L-NAME treatment. However, L-NAME induced bradycardia (n = 7-9 mice per group). (D) RSK2 expression in SM-specific RSK2 knockout (RSK2 SM -/- ) mice was reduced by approximately 87% following tamoxifen induction (n = 4-5 per group). (E) Myogenic responses of mesenteric resistance arteries from Y RSK2 +/+ (n = 4 mice, 8 arteries), A RSK2 +/+ (n = 4 mice, 8 arteries), and A RSK2 -/- (n = 4 mice, 8 arteries) at 80 mmHg. Myogenic tone was significantly greater in A RSK2 +/+ mice compared to Y RSK2 +/+ , but no difference was observed between Y RSK2 +/+ and A RSK2 -/- mice. (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The following antibodies were used: mouse monoclonal and rabbit polyclonal anti-actin (1:5,000 western blot (WB); Sigma-Aldrich); rabbit polyclonal antibodies anti-RSK2 phospho-Ser 227 and mouse monoclonal anti-RSK2 (1:500 WB; 1:50 IP, Santa Cruz Biotechnology Inc.); mouse monoclonal anti-AGE (1:50) antibody (MyBioSource); GAPDH (1:5,000 WB; Millipore).

Techniques: Western Blot, Expressing, Positive Control, Control, Knock-Out

Journal: bioRxiv

Article Title: Deletion of RSK2 kinase alleviates age-dependent hypertension

doi: 10.1101/2025.03.12.642932

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: mouse monoclonal and rabbit polyclonal anti-actin (1:5,000 western blot (WB); Sigma-Aldrich); rabbit polyclonal antibodies anti-RSK2 phospho-Ser 227 and mouse monoclonal anti-RSK2 (1:500 WB; 1:50 IP, Santa Cruz Biotechnology Inc.); mouse monoclonal anti-AGE (1:50) antibody (MyBioSource); GAPDH (1:5,000 WB; Millipore).

Techniques: Expressing

Journal: bioRxiv

Article Title: Deletion of RSK2 kinase alleviates age-dependent hypertension

doi: 10.1101/2025.03.12.642932

Figure Lengend Snippet: (A) Representative immunofluorescence images of en face second-order MAs from Y Rsk2 +/+ and A RSK2 +/+ mice, showing the organization of the internal elastic lamina (IEL). (B) Quantification of IEL characteristics: (left) total number of IEL holes within the field of view, (middle) total hole surface area, and (right) surface area normalized to the total number of IEL holes within the region of interest. A Rsk2 +/+ MAs exhibited a significantly lower number of IEL holes but a significantly larger IEL surface area compared to Y Rsk2 +/+ mice (n = 10). (C) Representative merged immunofluorescence images of en face preparations of second-order MAs from A Rsk2 +/+ and A Rsk2 -/- mice, showing IEL autofluorescence (green) and eNOS immunofluorescence (yellow). Black holes within the IEL represent MEJs. (D) Quantification of the percentage of eNOS immunofluorescence localized at MEPs ( n = 6). (E) Representative proximity ligation assay (PLA) merged images showing nuclei (blue) and white puncta indicating eNOS:Hbα co-localization in en face preparations of MAs from A Rsk2 +/+ and A Rsk2 -/- mice. (F) Quantification of eNOS:Hbα PLA puncta in MAs, normalized to nuclei ( n = 4-6). (*** P < 0.001).

Article Snippet: The following antibodies were used: mouse monoclonal and rabbit polyclonal anti-actin (1:5,000 western blot (WB); Sigma-Aldrich); rabbit polyclonal antibodies anti-RSK2 phospho-Ser 227 and mouse monoclonal anti-RSK2 (1:500 WB; 1:50 IP, Santa Cruz Biotechnology Inc.); mouse monoclonal anti-AGE (1:50) antibody (MyBioSource); GAPDH (1:5,000 WB; Millipore).

Techniques: Immunofluorescence, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Deletion of RSK2 kinase alleviates age-dependent hypertension

doi: 10.1101/2025.03.12.642932

Figure Lengend Snippet: (A-D) Representative images of mesenteric arterioles (MA) and iliac arteries stained with Masson’s trichrome, in young and aged mice. Lumen diameters were significantly larger in aged mesenteric arterioles and iliac arteries. Wall thickness did not differ in iliac arteries between groups; however, in A Rsk2 +/+ MAs, wall thickness was significantly greater compared to Y Rsk2 +/+ (P < 0.05). The ratio of wall area to total area between 20-month-old Rsk2 +/+ (n = 17) and Rsk2 -/- (n = 17) mice did not differ, suggesting that differences in cross-sectional dimensions reflect the overall larger size of aged mice compared to young mice . Aging did not increase vascular collagen content, as assessed by polarized light microscopy of picrosirius red-stained cross-sections of MAs and iliac arteries ( n = 11-26).

Article Snippet: The following antibodies were used: mouse monoclonal and rabbit polyclonal anti-actin (1:5,000 western blot (WB); Sigma-Aldrich); rabbit polyclonal antibodies anti-RSK2 phospho-Ser 227 and mouse monoclonal anti-RSK2 (1:500 WB; 1:50 IP, Santa Cruz Biotechnology Inc.); mouse monoclonal anti-AGE (1:50) antibody (MyBioSource); GAPDH (1:5,000 WB; Millipore).

Techniques: Staining, Light Microscopy

Journal: bioRxiv

Article Title: Deletion of RSK2 kinase alleviates age-dependent hypertension

doi: 10.1101/2025.03.12.642932

Figure Lengend Snippet: (A-C) Comparison of structural and mechanical parameters in MAs from young and aged Rsk2 +/+ and aged Rsk2 -/- mice. (A) Intraluminal diameter measurements of fully dilated MAs from A Rsk2 +/+ (n = 5), A Rsk2 -/- (n = 4), and Y Rsk2 +/+ (n = 6). (B) Wall-to-lumen ratio in MAs across all groups. (C) Stress-strain curves of fully dilated MAs. The stiffness coefficient (β) from the stress-strain relationship (inset) was significantly increased in both aged Rsk2 +/+ and aged Rsk2 -/- mice compared to young Rsk2 +/+ mice, with NS difference between the aged groups. (D) Advanced glycation end-product (AGE) crosslinking in iliac artery sections was significantly greater in aged Rsk2 +/+ and aged Rsk2 -/- mice compared to young Rsk2 +/+ . Secondary antibody-only reactions served as negative controls. Vessel media staining was blindly scored using the following criteria: 0 = grey/blue (no staining), 1 = light brown staining, 2 = light brown staining with some darker deposits, 3 = light brown staining with many darker deposits. Sample sizes: n = 4-6, Y Rsk2 +/+ : 10–15 weeks, A Rsk2 +/+ : 88– 91 weeks, A Rsk2 -/- : 88–90 weeks.

Article Snippet: The following antibodies were used: mouse monoclonal and rabbit polyclonal anti-actin (1:5,000 western blot (WB); Sigma-Aldrich); rabbit polyclonal antibodies anti-RSK2 phospho-Ser 227 and mouse monoclonal anti-RSK2 (1:500 WB; 1:50 IP, Santa Cruz Biotechnology Inc.); mouse monoclonal anti-AGE (1:50) antibody (MyBioSource); GAPDH (1:5,000 WB; Millipore).

Techniques: Comparison, Staining

Journal: bioRxiv

Article Title: Deletion of RSK2 kinase alleviates age-dependent hypertension

doi: 10.1101/2025.03.12.642932

Figure Lengend Snippet: Vascular aging and hypertension scheme: RSK2 shifts the balance of prorelaxant to procontractile mechanisms. In young arteries the balance between prorelaxant and procontractile mechanisms account for resting basal tone and normal BP. With aging, up regulation of RSK2 mediated procontractile and decreased prorelaxant mechanisms tip the balance to increased BP and hypertension.

Article Snippet: The following antibodies were used: mouse monoclonal and rabbit polyclonal anti-actin (1:5,000 western blot (WB); Sigma-Aldrich); rabbit polyclonal antibodies anti-RSK2 phospho-Ser 227 and mouse monoclonal anti-RSK2 (1:500 WB; 1:50 IP, Santa Cruz Biotechnology Inc.); mouse monoclonal anti-AGE (1:50) antibody (MyBioSource); GAPDH (1:5,000 WB; Millipore).

Techniques: