Journal: Cell stem cell

Article Title: Myoepithelial cells of submucosal glands can function as reserve stem cells to regenerate airways after injury

doi: 10.1016/j.stem.2018.03.018

Figure Lengend Snippet: TABLE FOR AUTHOR TO COMPLETE

Article Snippet: Y-27632 2HCl ROCK1 (p160ROCK inhibitor) , Selleckchem , S1049.

Techniques: Plasmid Preparation, Recombinant, Modification, Software

Journal: Investigative Ophthalmology & Visual Science

Article Title: The Intraocular Pressure Lowering Effect of a Dual Kinase Inhibitor (ITRI-E-(S)4046) in Ocular Hypertensive Animal Models

doi: 10.1167/iovs.62.13.12

Figure Lengend Snippet: The IC 50 for Kinases (ROCK, YSK4, and MYLK4), and pMLC2 were Examined After 72 Hours in HTM Cells for the Inhibitory Activity of ITRI-E-4046 Isomers

Article Snippet: Recombinant ROCK1 (Carna Biosciences, Kobe, Japan) was incubated with various concentrations of ROCK inhibitor in binding buffer (0.05 M Trizma hydrochloride buffer [pH 7.5] containing 0.1 M KCl, 0.01 M MgCl 2 , 0.1 mM EGTA, 30 mmol/L long S6 kinase substrate, and 1 mmol/L ATP) at room temperature for 90 minutes.

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of Na + /H + exchanger 1 specifically induces cell death in human iPS cells via sustained activation of the Rho kinase ROCK

doi: 10.1074/jbc.RA119.010329

Figure Lengend Snippet: ROCK accumulates in the plasma membrane upon overexpression of NHE1. A and B, subcellular localization of ROCK1 in untreated or Dox-treated iPSCs, respectively. ROCK1 was stained with the anti-ROCK1 antibody followed by an Alexa 488–conjugated secondary antibody, whereas NHE1 was stained with anti-HA followed by an Alexa 546–conjugated secondary antibody under nonpermeabilized conditions before ROCK1 staining. The slight intracellular red staining is due to fluorescence from mKate provided by the vector. C, cell fractionation was performed in Dox-treated or untreated iPSCs. Proteins (10 μg) from cytosolic and membrane fractions were subjected to immunoblot analysis. Antibodies against E-cadherin and GAPDH were used as membrane and cytosol markers, respectively. D, NHE1 was immunoprecipitated using HA-agarose beads, and then proteins were analyzed by immunoblotting using 4–12% gradient gel.

Article Snippet: Measurement of in vitro ROCK activity The recombinant MYPT1 (0.2 μg) was incubated at 37 °C with 5 ng of recombinant active ROCK1 (SignalChem) in 100 m m KCl, 10 m m MgCl 2 , 0.2 m m DTT, and mixed buffers (Mes, Mops, and Tris; 10 m m each) adjusted to various pH with KOH/HCl).

Techniques: Clinical Proteomics, Membrane, Over Expression, Staining, Fluorescence, Plasmid Preparation, Cell Fractionation, Western Blot, Immunoprecipitation

Journal: iScience

Article Title: Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

doi: 10.1016/j.isci.2024.110565

Figure Lengend Snippet: FAK translocation, ROCK remodulation, and activation of proliferation pathway (A) Estimation of uptake of α-SYN-TMR protofibrils by U251 cells treated with 1μM α-SYN protofibrils and pre-treated (before 30min) with 0.5 μM cytochalasin D and 5 μM Y-27632 (ROCK inhibitor) by flow cytometry. (B) Quantification of percentage gated fluorescence intensity of α-SYN-TMR protofibrils in the above experiment. (C) Fluorescence images of maximum intensity projected confocal z-stacks in astrocytes stained with Phalloidin (green) and FAK (red). The upper panel has nucleus stained with DAPI and the lower panel is without DAPI. White arrows indicate the nuclear colocalization of FAK, red arrows indicate TNTs and pink arrows indicate FAK at focal adhesion. (D) Quantification of FAK intensity in the nucleus of the cells with and without TNTs. (E and G) Fluorescence images of maximum intensity projected confocal z-stacks in U-87 MG cells stained with FAK (red) and pFAK (red) with DAPI, respectively. (F and H) Quantification of FAK and pFAK fluorescence intensity per cell in the nucleus. Quantifications are done from 15 image frames in a set, and each image frame has 15–20 cells. (I) Western blot images showing the change in ROCK1 and ROCK2 expressions with time on 1μM α-SYN protofibril treatment in U-87 MG cells. (J) Quantification of ROCK1 and ROCK2 western blots. Full-length western blots of four repeats are represented in Figure S9 . (K) Western blot images showing increased intensity of pERK1/pERK2, NF-κB, and Cdk1 with time on 1μM α-SYN protofibril treatment in U-87 MG cells. Full-length western blots of three repeats for pERK1/pERK2 and NF-κB and five repeats for Cdk1 were represented in Figure S10 . (L) Quantification of pERK1/pERK2, NF-κB, and Cdk1 western blots. Scale bars are denoted on the images. Data are expressed as mean ± SD, ∗∗∗ p ≤ 0.001. Statistics were analyzed using a two-way ANOVA. n = 3.

Article Snippet: Later the suspension was spun at 12,000 rpm for 10 min and the supernatant was collected and stored at -20 0 C. After normalizing the protein concentration, western blot assay was performed with primary antibodies GAPDH Mouse (Cloud-Clone # CAB932Hu22 dilution 1:1000), ROCK1 (CST # 4035T dilution 1:1000), ROCK2 (CST # 8236S dilution 1:1000), NF-kB p65/RelA Mouse mAb (Abclonal # A10609 dilution 1:1000), phospho ERK1-T202 + ERK2- T18 (Abclonal # AP0485 dilution 1:1000), Cdk1 Rabbit pAb (Abclonal #A0220 dilution 1:1000), p21 Waf1/Cip1 (12D1) Rabbit mAb (CST #2947 dilution 1:1000) and secondary antibodies, Goat anti-mouse (H+L) (Invitrogen #32430 dilution 1:1500) and Goat Anti-Rabbit (H+L) (Invitrogen #32460 1:1500).

Techniques: Translocation Assay, Activation Assay, Flow Cytometry, Fluorescence, Staining, Western Blot

Journal: iScience

Article Title: Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

doi: 10.1016/j.isci.2024.110565

Figure Lengend Snippet:

Article Snippet: Later the suspension was spun at 12,000 rpm for 10 min and the supernatant was collected and stored at -20 0 C. After normalizing the protein concentration, western blot assay was performed with primary antibodies GAPDH Mouse (Cloud-Clone # CAB932Hu22 dilution 1:1000), ROCK1 (CST # 4035T dilution 1:1000), ROCK2 (CST # 8236S dilution 1:1000), NF-kB p65/RelA Mouse mAb (Abclonal # A10609 dilution 1:1000), phospho ERK1-T202 + ERK2- T18 (Abclonal # AP0485 dilution 1:1000), Cdk1 Rabbit pAb (Abclonal #A0220 dilution 1:1000), p21 Waf1/Cip1 (12D1) Rabbit mAb (CST #2947 dilution 1:1000) and secondary antibodies, Goat anti-mouse (H+L) (Invitrogen #32430 dilution 1:1500) and Goat Anti-Rabbit (H+L) (Invitrogen #32460 1:1500).

Techniques: Recombinant, Staining, Software

Journal: Frontiers in Endocrinology

Article Title: PFKP Activation Ameliorates Foot Process Fusion in Podocytes in Diabetic Kidney Disease

doi: 10.3389/fendo.2021.797025

Figure Lengend Snippet: High glucose promoted PFKP expression and cytoskeletal remodeling in podocytes in vitro . (A) Representative western blots of PFKP expression in 30 mM HG-treated podocytes at various times points and quantification of these results, with α-tubulin as the loading control. (B) Representative western blots of PFKP expression in cultured podocytes stimulated with different concentrations of glucose for 24 h and quantification of these results, with α-tubulin set as the loading control. (C) Representative immunofluorescence staining of podocytes PFKP in each group. (D) PFK enzymatic activity in each group. (E) FBP level in each group. (F) ATP production in each group. (G) Western blotting analyses of the expression of ROCK1, PFKP and RhoA in podocytes in each group. GAPDH was set as the loading control. (H) Cytoskeletal structures of podocytes labeled with phalloidin. (I) Motility of podocytes quantified by a wound-healing assay using an inverted microscope. For all figures, n=3 independent experiments/group. * P < 0.05, ns, No significance. Scale bars: 10 µm. HG, high glucose; MA, mannitol; ROCK1, Rho-associated, coiled-coil containing protein kinase 1; PFKP, phosphofructokinase 1 platelet type; RhoA, RhoA-GTPase.

Article Snippet: The primary antibodies for Western blotting against ROCK1 (21850-1-AP, 1:1000), RhoA (10749-1-AP, 1:1000), α-tubulin (11224-1-AP, 1:1000), ALDOB (18065-1-AP,1:1000), and alpha-tubulin(11224-1-AP,1:1000) were purchased from proteintech (Wuhan, China).

Techniques: Expressing, In Vitro, Western Blot, Control, Cell Culture, Immunofluorescence, Staining, Activity Assay, Labeling, Wound Healing Assay, Inverted Microscopy

Journal: Frontiers in Endocrinology

Article Title: PFKP Activation Ameliorates Foot Process Fusion in Podocytes in Diabetic Kidney Disease

doi: 10.3389/fendo.2021.797025

Figure Lengend Snippet: Deletion of PFKP aggravated HG-induced cytoskeletal remodeling in podocytes in vitro . (A) Western blots of PFKP expression after transfection with various siPfkps , with GAPDH set as the loading control. (B) PFK enzymatic activity and FBP level in each group. (C) Cytoskeletal structures of podocytes labeled with phalloidin and the cytoskeletal rearrangement ratio in each group (original magnification ×600). (D) Western blotting analyses of the expression of ROCK1, PFKP, and RhoA in podocytes of each group. GAPDH was set as the loading control. (E) Motility of podocytes measured by a wound-healing assay using an inverted microscope. For all figures, n=3 independent experiments/group. * P < 0.05, ns, No significance. Scale bars: 10 µm. HG, high glucose; ROCK1, Rho-associated, coiled-coil containing protein kinase 1; PFKP, phosphofructokinase 1 platelet type; RhoA, RhoA-GTPase.

Article Snippet: The primary antibodies for Western blotting against ROCK1 (21850-1-AP, 1:1000), RhoA (10749-1-AP, 1:1000), α-tubulin (11224-1-AP, 1:1000), ALDOB (18065-1-AP,1:1000), and alpha-tubulin(11224-1-AP,1:1000) were purchased from proteintech (Wuhan, China).

Techniques: In Vitro, Western Blot, Expressing, Transfection, Control, Activity Assay, Labeling, Wound Healing Assay, Inverted Microscopy

Journal: Frontiers in Endocrinology

Article Title: PFKP Activation Ameliorates Foot Process Fusion in Podocytes in Diabetic Kidney Disease

doi: 10.3389/fendo.2021.797025

Figure Lengend Snippet: Overexpression of PFKP ameliorated HG-induced cytoskeletal remodeling in podocytes in vitro . (A) After recombinant plasmid (pcDNA3.1-Pfkp) transfection into podocytes, the expression of EGFP was observed by fluorescence microscopy. (B) Western blots of PFKP expression after transfection with pcDNA3.1-Pfkp, with GAPDH set as the loading control. (C) PFK enzymatic activity in each group. (D) FBP level in each group. (E) Western blotting analyses of the expression of ROCK1, PFKP, and RhoA in podocytes in each group. GAPDH was set as the loading control. (F) Cytoskeletal structures of podocytes labeled with phalloidin. (G) Motility of podocytes measured by a wound-healing assay using an inverted microscope. For all figures, n=3 independent experiments/group. * P < 0.05, ns, No significance. Scale bars: 10 µm. HG, high glucose; ROCK1, Rho-associated, coiled-coil containing protein kinase 1; PFKP, phosphofructokinase 1 platelet type; RhoA, RhoA-GTPase.

Article Snippet: The primary antibodies for Western blotting against ROCK1 (21850-1-AP, 1:1000), RhoA (10749-1-AP, 1:1000), α-tubulin (11224-1-AP, 1:1000), ALDOB (18065-1-AP,1:1000), and alpha-tubulin(11224-1-AP,1:1000) were purchased from proteintech (Wuhan, China).

Techniques: Over Expression, In Vitro, Recombinant, Plasmid Preparation, Transfection, Expressing, Fluorescence, Microscopy, Western Blot, Control, Activity Assay, Labeling, Wound Healing Assay, Inverted Microscopy

Journal: Frontiers in Endocrinology

Article Title: PFKP Activation Ameliorates Foot Process Fusion in Podocytes in Diabetic Kidney Disease

doi: 10.3389/fendo.2021.797025

Figure Lengend Snippet: FBP ameliorated HG-induced cytoskeletal remodeling in podocytes in vitro . (A) Targeted metabolomics was performed between control and HG group of cultured podocytes, and heat maps were generated based on levels of the metabolites related to glucose metabolism. (B) Western blots of ALDOB, ROCK1, PFKP, and RhoA after transfected with siAldob, GAPDH was set as the loading control. (C) FBP level in control and siAldob group. (D) Cytoskeletal structure of podocytes labeled with phalloidin in control and siAldob group. (E) Motility ability of podocytes were measured by wound-healing assay using an inverted microscope in control and siAldob group. (F) Western blotting analyses the expression of ALDOB, ROCK1, PFKP, and RhoA in podocytes after transfected with pcDNA3.1-Aldob. GAPDH was set as loading control. (G) FBP level in control and pcDNA3.1-Aldob group. (H) Cytoskeleton structure of podocytes labeled with phalloidin in control and pcDNA3.1-Aldob group. (I) Motility ability of podocytes were measured by wound-healing assay using an inverted microscope in control and pcDNA3.1-Aldob group. (J) Western blotting analyses the expression of ALDOB, ROCK1, PFKP, and RhoA in podocytes after FBP treatment. GAPDH was set as loading control. (K) Cytoskeleton structure of podocytes labeled with phalloidin in each group. (L) Motility ability of podocytes were measured by wound-healing assay using an inverted microscope in each group. For all figures, n=3 independent experiments/group. * P < 0.05, ns, No significance. Scale bars: 10 µm. HG, high glucose; FBP, fructose-1,6-bisphosphate; ROCK1, Rho-associated, coiled-coil containing protein kinase 1; PFKP, Phosphofructokinase 1 platelet type; RhoA, RhoA-GTPase.

Article Snippet: The primary antibodies for Western blotting against ROCK1 (21850-1-AP, 1:1000), RhoA (10749-1-AP, 1:1000), α-tubulin (11224-1-AP, 1:1000), ALDOB (18065-1-AP,1:1000), and alpha-tubulin(11224-1-AP,1:1000) were purchased from proteintech (Wuhan, China).

Techniques: In Vitro, Control, Cell Culture, Generated, Western Blot, Transfection, Labeling, Wound Healing Assay, Inverted Microscopy, Expressing