Journal: iScience

Article Title: Cocaine and habit training cause dendritic spine rearrangement in the prelimbic cortex

doi: 10.1016/j.isci.2023.106240

Figure Lengend Snippet:

Article Snippet: Rabbit anti p-ROCK2 , GeneTex , Cat. # GTX122651; RRID: AB_2560946.

Techniques: Virus, Plasmid Preparation, Recombinant, Software

Journal: JCI Insight

Article Title: ER stress and Rho kinase activation underlie the vasculopathy of CADASIL

doi: 10.1172/jci.insight.131344

Figure Lengend Snippet: ( A ) VSMC Rho kinase activity assessed by ELISA ( n = 4–5; Student’s t test). ( B and C ) Expression of ROCK2 ( B ) and ROCK1 ( C ) in CADASIL and control VSMCs in the absence and presence of a γ-secretase inhibitor (GSI). Upper panels: Representative immunoblots. Lower panels: Quantification of ROCK2 and ROCK1 protein expression normalized to α-tubulin ( n = 6–10; two-way ANOVA with Bonferroni’s post hoc test). ( D ) Calcium transients were measured by live cell fluorescence imaging using the fluoroprobe Cal-520 AM. Representative tracings of VSMC [Ca 2+ ] i responses to Ang II (1 × 10 –7 mol/L) in CADASIL and control groups. Experiments were repeated 4 times/group with greater than 30 cells studied/field. ( E ) [Ca 2+ ] i calculated as the area under the curve. Arrow indicates time of Ang II stimulation ( n = 4; Student’s t test). Results are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 versus control; # P < 0.05 versus vehicle plus CADASIL.

Article Snippet: Antibodies used were as follows: anti-ROCK2 (1:500, 610623, BD Biosciences); anti-ROCK1 (1:500; PIPA521130, Chemicon International); anti-PCNA (sc-56, Santa Cruz Biotechnology); anti-BiP (1:1000; 610978, BD Biosciences); anti-Notch3 (1:2000; 5276, Cell Signaling Technology); anti–β-actin (1:5000; A1978, Sigma-Aldrich); anti–α-tubulin (1:10000, ab4074, Abcam); anti–phospho-cofilin (3313S, Cell Signaling Technology); anti–phospho-vimentin (7391S, Cell Signaling Technology); anti-vimentin (3632S, Cell Signaling Technology); anti–phospho-filamin (4761S, Cell Signaling Technology); anti-filamin (4762S, Cell Signaling Technology); and anti-Nox5 (1: 1000; provided by David Harrison, Vanderbilt University Medical Center, Nashville, Tennessee, USA) ( ).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Fluorescence, Imaging

Journal: JCI Insight

Article Title: ER stress and Rho kinase activation underlie the vasculopathy of CADASIL

doi: 10.1172/jci.insight.131344

Figure Lengend Snippet: ( A – D ) Pretreatment of VSMCs with 4-PBA (1 × 10 –3 mol/L) or fasudil (1 × 10 –5 mol/L) reduced expression of ( A ) HEYL and ( B ) HES5 , and ER stress–related genes ( C ) AMFR6 and ( D ) EDEM1 in CADASIL VSMCs without effect on control VSMCs ( n = 3–14; one-way ANOVA with Bonferroni’s post hoc test). ( E ) Increased expression of BiP in CADASIL VMSCs was reduced in 4-PBA (1 × 10 –3 mol/L) and fasudil (1 × 10 –5 mol/L) pretreated cells. Upper panel: Representative immunoblot of BiP. BiP expression was normalized to β-actin ( n = 4–10; one-way ANOVA with Bonferroni’s post hoc test). ( F ) 4-PBA decreased Rho kinase activity in CADASIL VMSCs, as assessed by ELISA ( n = 4–12; one-way ANOVA with Bonferroni’s post hoc test). Results are expressed as mean ± SEM. * P < 0.05, ** P < 0.005 versus vehicle group from control VSMCs.

Article Snippet: Antibodies used were as follows: anti-ROCK2 (1:500, 610623, BD Biosciences); anti-ROCK1 (1:500; PIPA521130, Chemicon International); anti-PCNA (sc-56, Santa Cruz Biotechnology); anti-BiP (1:1000; 610978, BD Biosciences); anti-Notch3 (1:2000; 5276, Cell Signaling Technology); anti–β-actin (1:5000; A1978, Sigma-Aldrich); anti–α-tubulin (1:10000, ab4074, Abcam); anti–phospho-cofilin (3313S, Cell Signaling Technology); anti–phospho-vimentin (7391S, Cell Signaling Technology); anti-vimentin (3632S, Cell Signaling Technology); anti–phospho-filamin (4761S, Cell Signaling Technology); anti-filamin (4762S, Cell Signaling Technology); and anti-Nox5 (1: 1000; provided by David Harrison, Vanderbilt University Medical Center, Nashville, Tennessee, USA) ( ).

Techniques: Expressing, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Rho-associated kinase and zipper-interacting protein kinase, but not myosin light chain kinase, are involved in the regulation of myosin phosphorylation in serum-stimulated human arterial smooth muscle cells

doi: 10.1371/journal.pone.0226406

Figure Lengend Snippet: (A) Representative western blots showing the effects of ROCK1 or ZIPK knockdown on ROCK1 and ZIPK expression in CASMC and UASMC. (B) Quantification of ROCK1, ROCK2 and ZIPK levels in ROCK1 siRNA-treated cells compared to controls. (C) Quantification of ROCK1 and ZIPK levels in ZIPK siRNA-treated cells compared to controls. Statistical analysis was carried out with Dunnett’s post hoc test.

Article Snippet: The following primary antibodies were used in this study: rabbit monoclonal anti-ZIPK (1:1,000 dilution; Epitomics, Burlingame, CA; cat. no. 2568–1), rabbit polyclonal anti-ROCK1 (1:1,000 dilution; Epitomics; cat. no. S2711), rabbit polyclonal anti-ROCK2 (1:2,000 dilution; AbFrontier, Seoul, South Korea; cat. no. LF-PA0049), rabbit polyclonal anti-pT18/pS19-myosin light chain 2 (1:500 dilution; Cell Signaling; cat. no. 3674), rabbit anti-GAPDH (1:5,000 dilution; Santa Cruz Biotechnology; cat. no. sc-32233), goat polyclonal anti-SM22α (1:2,500 dilution; Novus Biologicals, Centennial, CO; cat. no. NB600-507), rabbit polyclonal anti-LC 20 (1:2,000 dilution; Rockland Immunochemicals, Limerick, PA; cat. no. 600-401-938), rabbit polyclonal anti-pT853-MYPT1 (1:1,000 dilution; EMD Millipore; cat. no. 36–003), rabbit polyclonal anti-pT696-MYPT1 (1:1,000 dilution; EMD Millipore; cat. no. ABS45), rabbit polyclonal anti-pT163-Par4 (1:1,000 dilution; Cell Signaling; cat. no. 2329), rabbit monoclonal anti-pS473-Akt (193H12) (1:1,000 dilution; Cell Signaling; cat. no. 4058); rabbit polyclonal anti-pT180/pY182-p38 MAP kinase (1:1,000 dilution; Cell Signaling; cat. no. 9211), rabbit monoclonal anti-pT202/Y204-ERK1/pT185/pY187-ERK2 (1:1,000 dilution; Cell Signaling; cat. no. 4370), rabbit anti-pS82-HSP27 (1:1,000 dilution; Cell Signaling; cat. no. 2401), and rabbit polyclonal anti-α-actin (1:1,000 dilution; Cytoskeleton, Inc.; cat. no. AAN01).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Rho-associated kinase and zipper-interacting protein kinase, but not myosin light chain kinase, are involved in the regulation of myosin phosphorylation in serum-stimulated human arterial smooth muscle cells

doi: 10.1371/journal.pone.0226406

Figure Lengend Snippet: A schematic showing the main conclusions from this study regarding serum-induced phosphorylation of the regulatory light chains of myosin II (LC 20 ) and the regulatory kinases and phosphatase involved. Serum treatment of CASMC and UASMC activates Rho-associated coiled-coil kinase (ROCK) and zipper-interacting protein kinase (ZIPK) leading to phosphorylation of the regulatory myosin-targeting subunit (MYPT1) of myosin light chain phosphatase (MLCP) at T853 and prostate apoptosis response 4 (Par4) at T163, both of which result in inhibition of MLCP activity. ZIPK activation involves phosphorylation by ROCK and autophosphorylation. Activated ROCK and ZIPK directly phosphorylate LC 20 at T18 and S19.

Article Snippet: The following primary antibodies were used in this study: rabbit monoclonal anti-ZIPK (1:1,000 dilution; Epitomics, Burlingame, CA; cat. no. 2568–1), rabbit polyclonal anti-ROCK1 (1:1,000 dilution; Epitomics; cat. no. S2711), rabbit polyclonal anti-ROCK2 (1:2,000 dilution; AbFrontier, Seoul, South Korea; cat. no. LF-PA0049), rabbit polyclonal anti-pT18/pS19-myosin light chain 2 (1:500 dilution; Cell Signaling; cat. no. 3674), rabbit anti-GAPDH (1:5,000 dilution; Santa Cruz Biotechnology; cat. no. sc-32233), goat polyclonal anti-SM22α (1:2,500 dilution; Novus Biologicals, Centennial, CO; cat. no. NB600-507), rabbit polyclonal anti-LC 20 (1:2,000 dilution; Rockland Immunochemicals, Limerick, PA; cat. no. 600-401-938), rabbit polyclonal anti-pT853-MYPT1 (1:1,000 dilution; EMD Millipore; cat. no. 36–003), rabbit polyclonal anti-pT696-MYPT1 (1:1,000 dilution; EMD Millipore; cat. no. ABS45), rabbit polyclonal anti-pT163-Par4 (1:1,000 dilution; Cell Signaling; cat. no. 2329), rabbit monoclonal anti-pS473-Akt (193H12) (1:1,000 dilution; Cell Signaling; cat. no. 4058); rabbit polyclonal anti-pT180/pY182-p38 MAP kinase (1:1,000 dilution; Cell Signaling; cat. no. 9211), rabbit monoclonal anti-pT202/Y204-ERK1/pT185/pY187-ERK2 (1:1,000 dilution; Cell Signaling; cat. no. 4370), rabbit anti-pS82-HSP27 (1:1,000 dilution; Cell Signaling; cat. no. 2401), and rabbit polyclonal anti-α-actin (1:1,000 dilution; Cytoskeleton, Inc.; cat. no. AAN01).

Techniques: Inhibition, Activity Assay, Activation Assay

Journal: iScience

Article Title: Broad and diverse roles of sphingosine-1-phosphate/sphingosine-1-phosphate receptors in the prostate

doi: 10.1016/j.isci.2024.111290

Figure Lengend Snippet: List of primary antibodies

Article Snippet: ROCK2 , Rabbit, monoclonal , – , 1:1000 , Abclonal, Cat. A2395.

Techniques: