phosphorylated erm  (Cell Signaling Technology Inc)

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD44/ERM/F‐actin complex mediates targeted nuclear degranulation and excessive neutrophil extracellular trap formation during sepsis

doi: 10.1111/jcmm.17231

Figure Lengend Snippet: CD44 translocation to the nucleus and delayed ‘targeted nuclear degranulation’ mediated by CD44/ERM/F‐actin. (A) Neutrophils were fluorescently stained for nuclear membrane, cell membrane and CD63, respectively. After stimulation of neutrophils with PMA (100 nM), CD63 co‐localization with the cell membrane (15 min) was earlier than CD63 co‐localization with the nuclear membrane (20 min) (scale bar: 5 μm). (B) PMA (100 nM) stimulates neutrophils for 0–20 min. Immunofluorescence staining of nuclear membrane (LaminB1,blue), skeleton (F‐actin, red) and CD44 (green), respectively. (C) PMA (100 nM) stimulates neutrophils for 0–20 min. WB for changes in nuclear membrane CD44, ERM and CD43 content changes. ERM phosphorylation inhibitor (P‐ERMi, 10 µM, 30 min) pre‐incubation of neutrophils for 30 min. WB detection of intracellular MPO, histone (H3) changes. (D) WB and ELISA for changes in intranuclear, cytoplasmic and extracellular CD44 content, flow cytometry for changes in cytosolic CD44 content. (E and F) P‐ERMi (10 µM, 30 min) pre‐incubation of neutrophils. (E) PMA (100 nM) stimulates neutrophils for 30 min, immunofluorescence staining for nuclear (DAPI,blue), P‐ERM (green) and CD44 (red). (F) PMA (100 nM) stimulates neutrophils for 120 min, SYTOX fluorescence staining. (G) Importini pre‐incubation of neutrophils, immunofluorescence staining of nuclear membrane (LaminB1,blue), skeleton (F‐actin, red) and CD44 (green). (H) Importini (30 µM) pre‐incubation of neutrophils, WB detection of nuclear membrane CD44 and changes in nuclear MPO content

Article Snippet: Wherever indicated, cells were preincubated with an ERM phosphorylation inhibitor (P‐ERMi) at 10 μM (MCE,HY‐18931A) or an importin‐β inhibitor (Importini) at 30 μM (MCE, HY‐101091) for 30 min before stimulation.

Techniques: Translocation Assay, Staining, Membrane, Immunofluorescence, Incubation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence

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: This membrane was blocked in 5 % skim milk in Tris buffered saline containing 0.1 % Tween-20 (TBS-T) and was incubated with primary antibodies overnight at 4°C — these primary antibodies were rabbit phosphorylated extracellular signal-regulated kinase (p-ERK), total ERK, ROCK1, ROCK2, phosphorylated ezrin-radixin-moesin (p-ERM), ERM (Cell Signaling Technology, Beverly, MA, USA), and mouse β-actin (Sigma).

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: This membrane was blocked in 5 % skim milk in Tris buffered saline containing 0.1 % Tween-20 (TBS-T) and was incubated with primary antibodies overnight at 4°C — these primary antibodies were rabbit phosphorylated extracellular signal-regulated kinase (p-ERK), total ERK, ROCK1, ROCK2, phosphorylated ezrin-radixin-moesin (p-ERM), ERM (Cell Signaling Technology, Beverly, MA, USA), and mouse β-actin (Sigma).

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: Hypothesis of signaling transduction mechanism of acupuncture analgesia from peripheral molecular responses to central analgesic pathway. Acupuncture was performed at the GB34 acupuncture point. In the local molecular changes, ERK was activated as a trigger molecule that activates ERM and ROCK proteins and the activated ROCK was strongly involved with acupuncture analgesia. Localized molecular signaling might be connected to the descending inhibitory pathway of supra-spinal tract, resulting in overall pain relief. Displacement, a linear movement of the needle; ERK, extracellular signal–regulated kinase; ERM, ezrin-radixin-moesin; Rev, revolution; ROCK, rho-associated protein kinase.

Article Snippet: This membrane was blocked in 5 % skim milk in Tris buffered saline containing 0.1 % Tween-20 (TBS-T) and was incubated with primary antibodies overnight at 4°C — these primary antibodies were rabbit phosphorylated extracellular signal-regulated kinase (p-ERK), total ERK, ROCK1, ROCK2, phosphorylated ezrin-radixin-moesin (p-ERM), ERM (Cell Signaling Technology, Beverly, MA, USA), and mouse β-actin (Sigma).

Techniques: Transduction

Journal: Veterinary and comparative oncology

Article Title: Protein kinase C regulates ezrin–radixin–moesin phosphorylation in canine osteosarcoma cells

doi: 10.1111/j.1476-5829.2010.00249.x

Figure Lengend Snippet: Ezrin (T567) phosphorylation is dependent on protein kinase C (PKC). (A) Canine osteosarcoma (OS) cells were incubated with various concentrations of PKC inhibitor Ro31–8220. Phospho-ERM expression is shown by western blot analysis (phosphorylated ERM is comprised of a phospho-ezrin/radixin band * and a phospho-moesin band **). (B) Canine OS cells were treated with PKC inhibitors Ro 31–8220 for indicated times. The level of phospho-ERM was analysed by Western blotting. The blots were probed for β-actin as the loading control, and phospho-Akt (Ser473).

Article Snippet: The membranes were blocked with 5% nonfat dried milk in TBS-Tween-20 (20 mmol/L Tris–HCl, pH 7.5, 8 g/L of sodium chloride, 0.1% Tween-20) and then incubated with anti-ezrin (1:4000 dilution) (Sigma, St Louis, MO, USA), anti-ERM (1:1000 dilution) (Cell Signaling, Beverly, MA, USA), anti-phosphorylated ERM (1:1000 dilution) (Cell Signaling, Beverly, MA, USA), anti-PKC α (1:1000 dilution) (Upstate, Swampscott, MA, USA), anti-PKC γ (1:1000 dilution) (BD Biosciences, Palo Alto, CA, USA), anti-PKC ι (1:250 dilution) (BD Biosciences, Palo Alto, CA, USA), anti-phosphorylated Akt (1:1000 dilution) (Cell Signaling, Beverly, MA, USA), anti-Akt (1:1000 dilution) (Cell Signaling, Beverly, MA, USA) or anit- β -actin (1:10 000 dilution) (Sigma, St Louis, MO, USA) overnight at 4 °C.

Techniques: Incubation, Expressing, Western Blot