Fig 2: Densitometric analysis of the proteins involved in Rho-Rac signaling at postnatal time-points in wild type (WT) and R6/2 mice. The three time-points represent different stages of disease progression in the R6/2 mouse: e.g. 3 week old (3w) = preclinical; 5w = emergence of motor abnormalities; and 10w = overt pathology. Densitometry was used to quantify the ratio of phosphorylated to total protein a ROCK2, b PAK, c LIMK1, d Cofilin, e SSH1L, f Profilin. Each value was initially normalized to expression of α-Tubulin in the corresponding sample. The data are presented as mean ± sem (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, between indicated groups

Fig 3: RND3 reduced HCC cell mobility partially by inhibiting ROCK1/2. (A and B): mRNA and protein expression level of indicated genes in HCC cells after RND3 knockdown, ROCK1 knockdown or ROCK2 knockdown. (C–F): Trans-well migration and invasion of HCC cells after different modifications and treatment. (G and H): MYPT1 protein expression level as well as its Thr853 phosphorylation (pT853) level in HCC cells in (C and D). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Fig 4: SUMOylation activates ROCK2 through PIAS1 E3 ligases.a, b Western analyses in 293T cells at 48 h post transfection with Myc-PIAS1, 2, 3, 4 or siRNAs of PIAS1, 2, 3, 4 in combination with or without Myc-SUMO1. c Western analyses in 293T cells at 48 h post transfection with or without Myc-PIAS1 and PIAS1 siRNA. d Co-immunoprecipitation experiments in 293T cells at 48 h post transfection with or without Myc-PIAS1 and Flag-ROCK2. e, f Western and co-immunoprecipitation analyses in 293T cells at 48 h post transfection with or without Myc-PIAS1 and Flag-ROCK2(WT or K1007R). g IHC of PIAS1 in human healthy bronchial sections (n = 4, left: experimental group, right: negative control group, scale bar, 2 μm). h, i Immunostaining of CC10, PIAS1, DAPI and semi-quantification in BALF cells from children with FBA or asthma (P value: <0.0001, scale bar, 10 μm). j–m Lungs or bronchi from NS- and OVA-challenged mice were subjected to IHC (j), qPCR (k, P value: 0.0127), and western analyses (l) and their semi-quantification (m, P value: 0.0093). Scale bar, 10 μm. n–q Mice were intratracheally instilled with lentiviral scramble- or PIAS1-shRNA and then with IL-13. Lungs and bronchi were subjected to H&E (scale bar, 10 μm), PAS (scale bar, 10 μm), Muc5AC (scale bar, 10 μm) and p-ROCK2 (scale bar, 5 μm) staining (n, o, P values: <0.0001, <0.0001; <0.0001, <0.0001; <0.0001, 0.0014) and western analyses (p, q, P values: <0.0001, 0.0002; 0.0002, 0.0031). Mean ± SD, n = 4, unpaired two-tailed Student’s t test or One-way ANOVA and Tukey-Kramer multiple comparisons test, *, +P < 0.05, **, ++P < 0.01. Experiments were repeated independently at least three times with similar results. Source data are provided as a Source Data file.

Fig 5: Pyr-apelin-13 enhanced the signaling pathways involved in cytoskeleton dynamics and proangiogenic actions in cultured endothelial cells exposed to high glucose conditions and hypoxia. BAECs were exposed to normal glucose (NG; 5.6 mmol/L; white bars) or high glucose (HG; 25 mmol/L; black bars) concentrations for 48 h and then stimulated with Pyr-apelin-13 for 1 h (A–C,E,F) or 24 h (D,G). BAECs were exposed to hypoxia (1% O2) for the last 16 h of treatment. Protein expression of (A) phospho-AMPKα1/2, (B) phospho-eNOS at Ser1177, (C) phospho-eNOS at Ser633, (E) phospho-ROCK-2 and (F) total protein expression of ROCK2 was detected by immunoblot analysis and the densitometry quantification was measured. mRNA levels of (D) eNOS and (G) RhoA. GAPDH gene was used for mRNA normalization. Results are presented as the mean ± SD of 8 (A) and 7 (B-G) independent cell experiments.