Journal: The FASEB Journal

Article Title: Cdc42 is involved in NC1 peptide–regulated BTB dynamics through actin and microtubule cytoskeletal reorganization

doi: 10.1096/fj.201900991R

Figure Lengend Snippet: Antibodies used in different experiments in this study

Article Snippet: Active RhoA-GTP (AB_1961799) , Mouse , NewEast Biosciences , 26904 , IP , .

Techniques:

Journal: The FASEB Journal

Article Title: Cdc42 is involved in NC1 peptide–regulated BTB dynamics through actin and microtubule cytoskeletal reorganization

doi: 10.1096/fj.201900991R

Figure Lengend Snippet: NC1-peptide–induced Sertoli cell TJ barrier disruption is mediated through Cdc42, which can be blocked by overexpression of Cdc42-T17N, a dominant negative mutant of Cdc42, in Sertoli cell epithelium. A) Regimens used for this study with Sertoli cells cultured in vitro at 0.4 × 106 cells/cm2 that formed an intact epithelium (see Materials and Methods) for different experiments in this report. B) A study by IB that confirmed overexpression of NC1 peptide following its overexpression in Sertoli cells without affecting the steady-state levels of Cdc42 or RhoA. β-Actin served as the protein loading control. Composite data of IB were shown on the right panel, wherein each bar is the mean ± sd of n = 3 experiments. Each experiment had triplicate cultures. **P < 0.01 by ANOVA. C) A pull-down assay was used to assess an activation of Cdc42 (i.e., GTP-bound Cdc42), but not RhoA, following overexpression of NC1 peptide in Sertoli cells with the corresponding negative (−ve) and positive (+ve) controls. N.s., not significantly different. Composite data of n = 3 experiments were shown on the right panel. **P < 0.01 by ANOVA. D) Overexpression of Cdc42-T17N mutant (a dominant negative mutant of Cdc42 by mutating residue Thr17 to Asn17) was confirmed by IB; however, its overexpression alone or cooverexpression with NC1 peptide did not alter the steady-state level of either NC1 peptide or Cdc42. The composite data of n = 3 independent experiments of this study were shown on the right panel. E) Overexpression of Cdc42-T17N mutant was able to eliminate the NC1 peptide–induced Cdc42 activation by quantifying the steady-state level of activated Cdc42 (i.e., Cdc432-GTP) by pull-down assays as noted by IB, including both −ve and +ve controls, with the composite data of n = 3 experiments in the lower panel. **P < 0.01 by ANOVA. F) A study by TER that quantified the Sertoli cell TJ permeability barrier function wherein overexpression of Cdc42-T17N mutant abolished the NC1 peptide–induced Sertoli cell TJ barrier disruption from a representative experiment with n = 4 bicameral units. Three additional experiments using different batches of Sertoli cells yielded similar results. **P < 0.01 by ANOVA by comparing to the other 3 groups. Uncropped blots corresponding to blots in B–E are shown in Supplemental Fig. S1. Ctrl, control.

Article Snippet: Active RhoA-GTP (AB_1961799) , Mouse , NewEast Biosciences , 26904 , IP , .

Techniques: Disruption, Over Expression, Dominant Negative Mutation, Cell Culture, In Vitro, Control, Pull Down Assay, Activation Assay, Mutagenesis, Residue, Permeability

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