Journal: bioRxiv

Article Title: Extracellular Pgk1 enhances neurite outgrowth of motoneurons through Nogo66-independent targeting of NogoA

doi: 10.1101/550921

Figure Lengend Snippet: The molecular pathway mediated by extracellular addition of Pgk1 to enhance neurite outgrowth is independent from the neuronal Nogo66/NgR Pathway. ( A ) Western blot analysis. Protein levels of phosphorylated ROCK2 at Y256 (p-ROCK2-Y256), ROCK2, phosphorylated LimK1 at T508 (p-Limk1-T508), LimK1, phosphorylated Cofilin at S3 (p-Cofilin-S3), total Cofilin, phosphorylated EGFR at Y1173 (p-EGFR-Y1173), EGFR, phosphorylated Akt at S473 (p-Akt-S473) and Akt in neurons cultured with Sol8-vector CM, Sol8-NogoA CM and Sol8-NogoA CM plus Pgk1 addition were all examined, as indicated. The α–tubulin served as internal loading control. ( B-F ) Quantification of protein expression level. The band intensities shown on Western blotting were quantified by ImageJ software. The relative value of each examined protein was used for comparison among the three groups when the value obtained from the Sol8-vector CM group was set as 1. All of the above data were averaged from three independent experiments. Statistical analysis used Student’s t -test (***, p <0.001; **, p <0.01; *, p <0.05).

Article Snippet: The antibodies against NogoA (SC; 1:500), Cofilin (Cell Signaling; CS; 1:1000), phosphorylated Cofilin at S3 (CS; 1:1000), Rho-associated protein kinase 2 (ROCK2) (CS; 1:2000), phosphorylated ROCK2 at Y256 (Rockland, 1:2000), Epidermal growth factor receptor (EGFR) (SC; 1:500), phosphorylated EGFR at Y1173 (CS; 1:2000), Akt (Protein kinase B; PKB) (CS; 1:1000), phosphorylated Akt at S473 (CS; 1:1000), LIM domain kinase 1 (Limk1; SC; 1:500), phosphorylated Limk1 at S323 (Signalway Antibody; SAB; 1:1000), phosphorylated Limk1 at T508 (SAB; 1:500), Phosphoglycerate kinase 1 (Pgk1; Abcam; 1:2000), p21-activated kinase 1 (Pak1) (CS; 1:1000), phosphorylated Pak1 at T423 (CS; 1:1000), P38 mitogen-activated protein kinases (P38) (CS; 1:1000), phosphorylated P38 at T180 (CS; 1:1000), MAP kinase-activated protein kinase 2 (MK2) (CS; 1:1000), phosphorylated MK2 at T334 (CS; 1:1000), Synapsin I (Syn1) (Abcam; 1:1000), Growth Associated Protein 43 (GAP43) (Abcam; 1:1000), Choline acetyltransferase (ChAT) (Abcam; 1:1000), Microtubule-associated protein 2 (MAP2) (Abcam; 1:1000), α-tubulin (SA; 1:5000), Myc (Sigma; 1:2000), Flag (Abcam; 1:5000), mouse-HRP (SC; 1:5000) and rabbit-HRP (SC; 1:5000) were used.

Techniques: Western Blot, Cell Culture, Plasmid Preparation, Expressing, Software

Journal: Integrative Medicine Research

Article Title: Peripheral Rho-associated protein kinase activation mediates acupuncture analgesia

doi: 10.1016/j.imr.2024.101051

Figure Lengend Snippet: ROCK1, ROCK2, and p-ERM expression in the skin after acupuncture treatment. Expression levels of (A) ROCK1, (B) ROCK2, and (C) p-ERM in the skin were determined at 5, 10, 30, and 60 min after acupuncture treatment via western blotting analyses. ROCK2 activation increased significantly at 30 and 60 min after acupuncture treatment compared to that in the control group (B). Activation of p-ERM was increased significantly 5 min after acupuncture treatment (C). Here, * p < 0.05 compared to the CON group followed by the Student's t -test (each n = 3–5) and error bars indicate the SEM. CON, treated with no acupuncture; ROCK, Rho-associated protein kinase; p-ERM, phosphorylated ezrin-radixin-moesin.

Article Snippet: For immunohistochemistry analyses, all sections were incubated in 3 % hydrogen peroxide for 10 min, washed in wash buffer (TBS-T), and were incubated in blocking solution (i.e., TBS-T with 5 % BSA) for 1 h. The blocking solution was removed and the primary rabbit ROCK2 antibody (Novus Biologicals, Littleton, CO, USA) was diluted in the blocking solution and were added to each section; these solutions were incubated overnight at 4°C.

Techniques: Expressing, Western Blot, Activation Assay, Control

Journal: Integrative Medicine Research

Article Title: Peripheral Rho-associated protein kinase activation mediates acupuncture analgesia

doi: 10.1016/j.imr.2024.101051

Figure Lengend Snippet: ROCK2 expression in the skin after acupuncture treatment, and inhibition of acupuncture-induced ROCK2, p-ERM, and p-ERK activation by U0126 (i.e., ERK inhibitor) and Y-27632 (i.e., ROCK inhibitor). (A-B) ROCK2 was activated in the skin, especially in the fibroblasts of the dermis 30 min after acupuncture treatment. 5B5, fibroblast marker. Blue: counterstain with 40,6-diamidino-2-phenylindole (DAPI), Green: expression of ROCK2, Red: expression of 5B5, a fibroblast marker. (C-D) Acupuncture induced ROCK2 and p-ERM activation was attenuated by U0126 administration, injected at the GB34 acupuncture point intradermally. (E) Acupuncture-induced ROCK2 activation in the skin was significantly attenuated by 0.3 μg/ul of Y-27632. (F) Acupuncture induced p-ERK expression was attenuated by U0126, but not by Y-27632 administration. Acupuncture induced p-ERM expression was attenuated by U0126 and Y-27632 administration. Scale bar: 100 µm (A) and 20 µm (B). ∗∗ p < 0.01 compared to the CON group; $$ p < 0.01 compared to the ACU group followed by the Student's t -test (each n = 3). Error bars indicate the SEM. ACU, treated with acupuncture; ACU+U, treated with U0126 and acupuncture; ACU+Y, treated with Y-27632 and acupuncture; CON, treated with no acupuncture; p-ERK, phosphorylated extracellular signal-regulated kinase; p-ERM, phosphorylated ezrin-radixin-moesin; ROCK, Rho-associated protein kinase.

Article Snippet: For immunohistochemistry analyses, all sections were incubated in 3 % hydrogen peroxide for 10 min, washed in wash buffer (TBS-T), and were incubated in blocking solution (i.e., TBS-T with 5 % BSA) for 1 h. The blocking solution was removed and the primary rabbit ROCK2 antibody (Novus Biologicals, Littleton, CO, USA) was diluted in the blocking solution and were added to each section; these solutions were incubated overnight at 4°C.

Techniques: Expressing, Inhibition, Activation Assay, Marker, Injection

Journal: Integrative Medicine Research

Article Title: Peripheral Rho-associated protein kinase activation mediates acupuncture analgesia

doi: 10.1016/j.imr.2024.101051

Figure Lengend Snippet: ROCK1, ROCK2, and p-ERM expression in the skin after acupuncture treatment. Expression levels of (A) ROCK1, (B) ROCK2, and (C) p-ERM in the skin were determined at 5, 10, 30, and 60 min after acupuncture treatment via western blotting analyses. ROCK2 activation increased significantly at 30 and 60 min after acupuncture treatment compared to that in the control group (B). Activation of p-ERM was increased significantly 5 min after acupuncture treatment (C). Here, * p < 0.05 compared to the CON group followed by the Student's t -test (each n = 3–5) and error bars indicate the SEM. CON, treated with no acupuncture; ROCK, Rho-associated protein kinase; p-ERM, phosphorylated ezrin-radixin-moesin.

Article Snippet: For immunofluorescence analyses, all sections were washed in a wash buffer and then incubated in blocking solution for 1 h. The blocking solution was removed and the primary antibodies of ROCK2 (Novus Biologicals) and mouse fibroblasts (5B5; Abcam, Cambridge, United Kingdom) were diluted in the blocking solution and were added to the sections; these solutions were incubated overnight at 4°C.

Techniques: Expressing, Western Blot, Activation Assay, Control

Journal: Integrative Medicine Research

Article Title: Peripheral Rho-associated protein kinase activation mediates acupuncture analgesia

doi: 10.1016/j.imr.2024.101051

Figure Lengend Snippet: ROCK2 expression in the skin after acupuncture treatment, and inhibition of acupuncture-induced ROCK2, p-ERM, and p-ERK activation by U0126 (i.e., ERK inhibitor) and Y-27632 (i.e., ROCK inhibitor). (A-B) ROCK2 was activated in the skin, especially in the fibroblasts of the dermis 30 min after acupuncture treatment. 5B5, fibroblast marker. Blue: counterstain with 40,6-diamidino-2-phenylindole (DAPI), Green: expression of ROCK2, Red: expression of 5B5, a fibroblast marker. (C-D) Acupuncture induced ROCK2 and p-ERM activation was attenuated by U0126 administration, injected at the GB34 acupuncture point intradermally. (E) Acupuncture-induced ROCK2 activation in the skin was significantly attenuated by 0.3 μg/ul of Y-27632. (F) Acupuncture induced p-ERK expression was attenuated by U0126, but not by Y-27632 administration. Acupuncture induced p-ERM expression was attenuated by U0126 and Y-27632 administration. Scale bar: 100 µm (A) and 20 µm (B). ∗∗ p < 0.01 compared to the CON group; $$ p < 0.01 compared to the ACU group followed by the Student's t -test (each n = 3). Error bars indicate the SEM. ACU, treated with acupuncture; ACU+U, treated with U0126 and acupuncture; ACU+Y, treated with Y-27632 and acupuncture; CON, treated with no acupuncture; p-ERK, phosphorylated extracellular signal-regulated kinase; p-ERM, phosphorylated ezrin-radixin-moesin; ROCK, Rho-associated protein kinase.

Article Snippet: For immunofluorescence analyses, all sections were washed in a wash buffer and then incubated in blocking solution for 1 h. The blocking solution was removed and the primary antibodies of ROCK2 (Novus Biologicals) and mouse fibroblasts (5B5; Abcam, Cambridge, United Kingdom) were diluted in the blocking solution and were added to the sections; these solutions were incubated overnight at 4°C.

Techniques: Expressing, Inhibition, Activation Assay, Marker, Injection

Journal: Scientific Reports

Article Title: Activated ROCK/Akt/eNOS and ET-1/ERK pathways in 5-fluorouracil-induced cardiotoxicity: modulation by simvastatin

doi: 10.1038/s41598-020-71531-8

Figure Lengend Snippet: Effect of 5-FU and Sim treatments on cardiac ( A ) content of p -ERK, ( B ) expression of ROCK, ( C ) expression and ( D ) activity of caspase-3 and ( E ) DNA fragmentation analysis, in which 5-FU group shows DNA laddering. Values are presented as the mean of 6–8 experiments ± SEM. Statistical analysis was carried out using one-way ANOVA followed by Tukey’s post- hoc test ; as compared to normal (*), Sim (#), and 5-FU (@)-treated groups (p < 0.05). 5-FU 5-fluorouracil, M , marker DNA (1 kb), p-ERK1/2 (Thr202/Tyr204) , phosphorylated extracellular signal-regulated kinase 1/2 at Thr 202 & Tyr 204, ROCK rho-kinase, Sim simvastatin.

Article Snippet: The protein expression was assessed as previously described by Ahmed et al . . Concisely, the membranes were incubated overnight with primary antibodies against ROCK (1:1,000, R&D systems, MN, USA; Cat. # AF4790-SP), caspase-3 (1:1,000, MyBioSource, CA, USA; Cat. # MBS9382732) or β-actin (1:1,000, ThermoFisher Scientific, MA, USA; Cat. # PA1-183).

Techniques: Expressing, Activity Assay, DNA Laddering, Marker