Journal: bioRxiv

Article Title: Astrocytes close the critical period for visual plasticity

doi: 10.1101/2020.09.30.321497

Figure Lengend Snippet: (a) Top, volcano plot analysis representing the 2096 quantified proteins with at least 3 total peptides in all replicates enriched with biotinylated WFA lectin in KD and WT mice. Binding partners were obtained by using quantitative label-free mass spectrometry analysis performed from three replicates. Dashed vertical lines denote absolute fold change of 1.5 and the dashed horizontal line denotes the adjusted P-value of ratio significance of 0.05. Selected enriched proteins in WT (green) and KD (purple) samples are shown. Proteins from the enriched pathways Rho-GTPase activate KTN1 (blue) and Rho-GTPase activate ROCK (red) are highlighted. External plots show proteins with peptides identified only in one sample type (left in KD and right in WT). Bottom, volcano plot analysis representing interactors of Cx30 from the Rho-GTPase activate KTN1 (blue) and Rho-GTPase activate ROCK (red) pathways. The fold-change in WT (n=5) versus KD mice (n=4) are shown with selected proteins (Rock2 and Myosin 14) quantified with an absolute fold change ≥ 1.5, an adjusted P-value ≤ 0.05 and with ≥ 3 peptides. (b) Immunostaining for RhoA-GTP showed a marked increase in KD (n=27, P=0.0003), KD MD (n=16, P<0.0001) and WT MD mice (n=16, P<0.0001) compared to WT mice (n=22) (slices from 3 mice per group) (Kruskal-Wallis (KW=41.23) followed by Dunn’s post-hoc test. (c) Immunostaining for MMP9 shows a marked increase in KD mice (n=28, P<0.0001, DF=71), KD MD (n=13, P=0.0059, DF=71) and MD mice (n=12, P=0.0322, DF=71) compared to WT mice (n=22) (ANOVA (F(3,71)=15.07) followed by Tukey’s post-hoc test). (d) Diagram depicting the protocol for experimental treatment with Fasudil. (e) Fasudil rescued MMP9 levels in KD mice (KD+Fasudil: n=25 from 5 mice, KD=28 slices from 3 mice, P<0.0001), while it had no effect in WT mice (WT+Fasudil: n=34 from 6 mice, WT: 22 slices from 3 mice, P>0.9999, Kruskall-Wallis (KW=55.98) followed by Dunn’s post-hoc test). (f) Fasudil rescued perineuronal nets levels in KD mice (KD+Fasudil: n=52 from 5 mice; KD, n=177 from 16 mice, P<0.0001) while it had no effect in WT mice (WT+Fasudil, n=129 from 6 mice, WT, n=226 from 16 mice, P=0.1510) (Kruskall-Wallis (KW=71.65) followed by Dunn’s post-hoc test).

Article Snippet: The following primary antibodies were used: Cx30 rabbit polyclonal (1:500, 71-2200, Zymed), Cx43 mouse monoclonal (1:500, 610061 BD Biosciences), GFAP mouse monoclonal (1:500, G3893, Sigma-Aldrich), chick anti GFP (1:500, AB13970, Abcam), Parvalbumin mouse monoclonal (1:500, 235, SWANT), MMP9 rabbit (1:500, 3852, cell signaling), anti-active RhoA mouse monoclonal (1:500, 26904, NewEast Biosciences), Lectin from Wisteria Floribunda, biotin conjugate (1:500, L1516-2MG, Sigma-Aldrich).

Techniques: Binding Assay, Mass Spectrometry, Immunostaining

Journal: Molecular neurobiology

Article Title: Semaphorin3F Drives Dendritic Spine Pruning through Rho-GTPase Signaling

doi: 10.1007/s12035-021-02373-2

Figure Lengend Snippet: Postulated Dual Signaling Pathway for Sema3F-induced Spine Pruning through Rac1 and RhoA.

Article Snippet: Filters were blocked in 5% nonfat milk in TBS-Tween 20 (0.1%) and incubated overnight with rabbit anti-NrCAM (R&D Systems AF8538, 1:1000), mouse anti-Rac1 (BD Biosciences # 610651, RRID:AB_397978, 1:1000), mouse anti-RhoA (BD Transduction Laboratories #610990, 1:500), or mouse anti-Cdc42 (MAb28-10).

Techniques:

Journal: Molecular neurobiology

Article Title: Semaphorin3F Drives Dendritic Spine Pruning through Rho-GTPase Signaling

doi: 10.1007/s12035-021-02373-2

Figure Lengend Snippet: Sema3F-induced spine remodeling in cortical neuron cultures is mediated through Rac1 and RhoA GTPase.

Article Snippet: Filters were blocked in 5% nonfat milk in TBS-Tween 20 (0.1%) and incubated overnight with rabbit anti-NrCAM (R&D Systems AF8538, 1:1000), mouse anti-Rac1 (BD Biosciences # 610651, RRID:AB_397978, 1:1000), mouse anti-RhoA (BD Transduction Laboratories #610990, 1:500), or mouse anti-Cdc42 (MAb28-10).

Techniques:

Journal: Basic Research in Cardiology

Article Title: RGS3L allows for an M 2 muscarinic receptor-mediated RhoA-dependent inotropy in cardiomyocytes

doi: 10.1007/s00395-022-00915-w

Figure Lengend Snippet: Effect of the expression level of RGS3L on the carbachol-induced RhoGTPase activation in NRCM. A Identification of the specific RGS3 isoform expressed in NRCMs treated under serum-free conditions with or without 50 ng/ml FGF2 for 24 h, as indicated. RGS3 in the cell lysates was analyzed by 2D gel electrophoresis and immunoblot. G β was used as a loading control ( B , C ). Measurement of the carbachol-induced RhoA ( B ) and Rac1 activation ( C ) in NRCMs without or with FGF2 treatment stimulated without and with 1 mM carbachol for 5 min. Levels of Rac1–GTP and RhoA–GTP were measured by Rac1 and RhoA activation assays. Quantification of Rac1 (Rac–GTP/total Rac1) and RhoA activity (RhoA–GTP/total RhoA) as well as a representative experiment are shown. The mean of Rac1–GTP or RhoA–GTP levels detected in unstimulated cells was set to 1.0. Values are mean + SD ( n = 8/5 RhoA/Rac1), * p < 0.05. D , E NRMCs were transduced with Ad-EGFP (Control, MOI 40) and increasing amounts (MOI 10, 20, 30) of Ad-RGS3L–N460A for 48 h. Levels of RhoA–GTP ( D ) and Rac1–GTP ( E ) were determined after 5 min stimulation without or with carbachol. Recombinant RGS3L expression was monitored by immunoblot analysis using the anti-c-myc antibody. F , G NRCMs were transduced with Ad-shEGFP and Ad-shRGS3L for 72 h. RGS3L expression was monitored by 2D gel electrophoresis and immunoblot. Gβ was used as a loading control. F Cells were incubated for 5 min with or without carbachol. Levels of RhoA–GTP and Rac1–GTP were determined

Article Snippet: Laboratories, 610149), mouse-anti-RhoA (26C4, Santa Cruz, sc-418), mouse–anti-RGS3 (CC-Q7, Santa Cruz, sc-100762), rabbit–anti-Tiam1 (C-16, Santa Cruz, sc-872), rabbit–anti-Gβ (T-20, Santa Cruz, sc-378), rabbit–anti-RGS3 (Abcam, ab2564), mouse–anti-c-myc (Klon 9E10, Oncogene), mouse–anti-β-actin (Sigma-Aldrich, A2228), mouse–anti-RhoA-GTP (Biomol).

Techniques: Expressing, Activation Assay, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Activity Assay, Transduction, Recombinant, Incubation

Journal: Basic Research in Cardiology

Article Title: RGS3L allows for an M 2 muscarinic receptor-mediated RhoA-dependent inotropy in cardiomyocytes

doi: 10.1007/s00395-022-00915-w

Figure Lengend Snippet: Effect of RGS3L–N460A expression on the M 2 R-mediated RhoA activation in AMVCM. Cells were transduced with Ad-RGS3L–N460A or Ad-EGFP for 48 h. Cells were stimulated with or without carbachol for 5 min. RhoA–GTP was detected with mouse–anti-RhoA–GTP-antibody and secondary anti-mouse-Alexa Fluor ® 568-antibody, RGS3L–N460A was detected using rabbit–anti-c-myc antibody and a secondary anti-rabbit-Alexa Fluor ® 633-antibody. DAPI was used to detect nuclei. Sarcomeric proteins are visualized by a non-specific incorporation of EGFP. Cells were visualized by confocal microscopy (Leica, Germany) and fluorescence at the sarcolemma was analyzed using ImageJ-software. A , B Localization of RhoA–GTP and RGS3L–N460A in AMVCM transduced with RGS3L–N460A adenovirus and stimulated with carbachol. Visualization of RhoA–GTP in representative cells ( C ) and analysis of the fluorescence intensity of the Alexa Fluor ® 568 conjugate (which corresponds to the RhoA–GTP-amount) at the sarcolemma in 5 transductions obtained from 3 independent AMVCM preparations (D ). Values are mean + SD, * p < 0.05

Article Snippet: Laboratories, 610149), mouse-anti-RhoA (26C4, Santa Cruz, sc-418), mouse–anti-RGS3 (CC-Q7, Santa Cruz, sc-100762), rabbit–anti-Tiam1 (C-16, Santa Cruz, sc-872), rabbit–anti-Gβ (T-20, Santa Cruz, sc-378), rabbit–anti-RGS3 (Abcam, ab2564), mouse–anti-c-myc (Klon 9E10, Oncogene), mouse–anti-β-actin (Sigma-Aldrich, A2228), mouse–anti-RhoA-GTP (Biomol).

Techniques: Expressing, Activation Assay, Transduction, Confocal Microscopy, Fluorescence, Software

Journal: Basic Research in Cardiology

Article Title: RGS3L allows for an M 2 muscarinic receptor-mediated RhoA-dependent inotropy in cardiomyocytes

doi: 10.1007/s00395-022-00915-w

Figure Lengend Snippet: Effect of Tiam1-downregulation and RGS3L–N460A overexpression on carbachol-induced Rac1-activation and Rac1-effector functions NRCMs. Cells were transduced with or without Ad-shEGFP or Ad-shTiam1 for 72 h. A Tiam1 expression was monitored by 2D gel electrophoresis and immunoblot. Gβ was used as a loading control. B Cells were transduced with Ad-shEGFP, Ad-shTiam1 and Ad-RGS3L–N460A for 72 h as indicated. Levels of RhoA–GTP and Rac1–GTP were determined after 5 min stimulation with or without carbachol. C , D Quantification of ROS-production in NRCMs by DCF fluorescence. C NRCMs were transduced with Ad-shEGFP or Ad-shTiam1 for 72 h. D Cells were transduced with Ad-EGFP or Ad-RGS3L–N460A for 24 h and additionally treated with 100 ng/ml PTX or 50 ng/ml FGF2 for 24 h as indicated. Thereafter, the cells were stimulated with carbachol or solvent for 5 min at 37 °C. Values are mean + SD ( n = 6–10), * p < 0.05. E Effect of the RGS3L–N460A-Expression on the Phenylephrine (PE) induced cellular hypertrophy. One μCi/ml [ 3 H]–leucine was added into the culture medium shortly after stimulation. After 24 h cellular protein was collected and [ 3 H]-content was determined. Measurements were performed in 3 replicates. Values are mean + SD ( n = 3/6 (EGFP/RGS3L–N460A), * p < 0.05

Article Snippet: Laboratories, 610149), mouse-anti-RhoA (26C4, Santa Cruz, sc-418), mouse–anti-RGS3 (CC-Q7, Santa Cruz, sc-100762), rabbit–anti-Tiam1 (C-16, Santa Cruz, sc-872), rabbit–anti-Gβ (T-20, Santa Cruz, sc-378), rabbit–anti-RGS3 (Abcam, ab2564), mouse–anti-c-myc (Klon 9E10, Oncogene), mouse–anti-β-actin (Sigma-Aldrich, A2228), mouse–anti-RhoA-GTP (Biomol).

Techniques: Over Expression, Activation Assay, Transduction, Expressing, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Fluorescence