anti cdh6  (R&D Systems)

Journal: The FEBS journal

Article Title: Cadherin-6 controls neuronal migration during mouse neocortical development via an integrin-mediated pathway.

doi: 10.1111/febs.70150

Figure Lengend Snippet: Fig. 1. CDH6 protein is detected in the developing mouse cerebral cortex. (A–G) Immunostaining for CDH6 (green) and DAPI (magenta) using sections from the cerebral cortex at E12.0 (A) (n = 6), E14.0 (B) (n = 6), E16.0 (C) (n = 4) and E18.0 (D–F) (n = 10) of the ICR mouse. The boxed regions in (D) are shown at higher magnification in (E) and (F). (G) Control staining with normal sheep IgG instead of CDH6 antibody (n = 10). The lower panels in (A–G) show grayscale, single-channel images of CDH6 (A–F) and normal sheep IgG (G), respectively. (H–J) The cerebral cortices (E17.0) that had been electroporated with CAG-EGFP plasmid on E14.0 were stained for DCX (H, I) (n = 4) and Nestin (NES) (n = 4) (J). Arrows in (H) and (I) indicate colocalization of immunosignals of CDH6/DCX in EGFP-positive neurons in CP and IZ, respectively. Cell outline is highlighted using the GFP signal as a guide in the CDH6 and DCX panels (magenta lines in H and I). The second panels from the left in (H–J) show CDH6 grayscale, single-channel images. The fourth panels from the left in (H–I) show DCX grayscale, single-channel images. The fourth panel from the left in (J) shows a NES grayscale, single-channel image. (K–M) Immunostaining for HA (green) and DAPI (magenta) using sections from the cerebral cortex at P0 of the Cdh6-HA KI mouse (n = 4). Boxed regions in (K) are shown at higher magnification in (L) and (M). (N) Control staining with normal rat IgG instead of HA antibody (n = 4). The lower panels in (K–N) show grayscale, single-channel images of CDH6 (K–M) and normal rat IgG (N), respectively. Scale bars: 100 lm in (A, B, C, D, G, K, N); 40 lm in (E, F, L, M); 5 lm in (H, I); 20 lm in (J). CP, cortical plate; DCX, Doublecortin; HP, hippocampus; IZ, intermediate zone; MAZ, multipolar cell accumulation zone; MZ, marginal zone; Ncx, neocortex; NES, Nestin; SLM, stratum lacunosum moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; VZ, ventricular zone.

Article Snippet: The following primary antibodies were used in this study: CDH6 (sheep; R&D Systems, AF2715, 1 : 200), HA (rat; Roche, 11867423001, 1 : 1500), GAPDH (mouse; Santa Cruz Biotechnology, Dallas, TX, USA, sc-32233, 1 : 1000), integrin b1 (rabbit; Abcam, ab183666, 1 : 1200), b-Actin (mouse; Sigma-Aldrich, MO, USA, A5441, 1 : 1000), Paxillin (rabbit; Abcam, ab32084, 1 : 1000), pFAK (Y397) (rabbit; CST, 3283S, 1 : 1500), and FAK (mouse; BD, 610088, 1 : 1000).

Techniques: Immunostaining, Control, Staining, Plasmid Preparation

Journal: The FEBS journal

Article Title: Cadherin-6 controls neuronal migration during mouse neocortical development via an integrin-mediated pathway.

doi: 10.1111/febs.70150

Figure Lengend Snippet: Fig. 3. CDH6 is involved in the proper neuronal motility. (A) Time-lapse images of control and Cdh6 KD neurons migrating in the IZ, taken at 10-min intervals. Images taken every 40 min are shown. Arrows indicate representative tracked neurons. (B) Migration speed was slower in the Cdh6 KD case than in the control case (control, 32.0 2.9 lmh1, n = 16 cells from 3 mice; Cdh6 KD, 25.1 1.7 lmh1, n = 18 cells from 3 mice; P* = 0.0361) (Student’s t-test). Data are mean SEM. (C) E14.0 embryos were electroporated with the control or Cdh6 KD vector and CAG-EGFP, and the length and orientation of leading processes of EGFP-labeled neurons migrating in the superficial part of the IZ and CP were examined at E17.0. (D) The length of leading process was not altered in the Cdh6 KD case compared with the control case (control, 21.3 0.6 lm, n = 114 cells from 4 mice; Cdh6 KD, 20.3 0.5 lm, n = 96 cells from 4 mice; P = 0.17) (Student’s t-test). Data are mean SEM. (E) The angle of the leading process was not changed in the Cdh6 KD case compared with the control case (control, 7.4 0.7 lmh1, n = 114 cells from 4 mice; Cdh6 KD, 7.7 0.6, n = 96 cells from 4 mice; P = 0.67) (Student’s t-test). Data are mean SEM. Scale bars: 50 lm in (A), 30 lm in (C). CP, cortical plate; IZ, intermediate zone; KD, knockdown.

Article Snippet: The following primary antibodies were used in this study: CDH6 (sheep; R&D Systems, AF2715, 1 : 200), HA (rat; Roche, 11867423001, 1 : 1500), GAPDH (mouse; Santa Cruz Biotechnology, Dallas, TX, USA, sc-32233, 1 : 1000), integrin b1 (rabbit; Abcam, ab183666, 1 : 1200), b-Actin (mouse; Sigma-Aldrich, MO, USA, A5441, 1 : 1000), Paxillin (rabbit; Abcam, ab32084, 1 : 1000), pFAK (Y397) (rabbit; CST, 3283S, 1 : 1500), and FAK (mouse; BD, 610088, 1 : 1000).

Techniques: Control, Migration, Plasmid Preparation, Labeling, Knockdown

Journal: The FEBS journal

Article Title: Cadherin-6 controls neuronal migration during mouse neocortical development via an integrin-mediated pathway.

doi: 10.1111/febs.70150

Figure Lengend Snippet: Fig. 5. CDH6 regulates neuronal migration in the hippocampal CA1 in an RGD motif-dependent manner. (A) The CA1 regions that had been electroporated with control or Cdh6 KD vector with CAG- EGFP plasmid at E14.0 were analyzed at E18.0. (B) Graphs show quantification of cell migration in (A) (control, n = 4; Cdh6 KD, n = 10; Cdh6 KD + Cdh6 WT*, n = 5; Cdh6 KD + Cdh6 RGD mut*, n = 5). Data are mean SEM. Statistically significant differences were observed in control vs. Cdh6 KD (***P < 0.001), control vs. Cdh6 KD + Cdh6 RGD mut* (***P < 0.001), Cdh6 KD vs. Cdh6 KD + Cdh6 WT* (*P = 0.025), and Cdh6 KD + Cdh6 WT* vs. Cdh6 KD + Cdh6 RGD mut* (*P = 0.044) (Tukey–Kramer test). Scale bar: 50 lm in (A). KD, knockdown; mut, mutant; WT, wild-type.

Article Snippet: The following primary antibodies were used in this study: CDH6 (sheep; R&D Systems, AF2715, 1 : 200), HA (rat; Roche, 11867423001, 1 : 1500), GAPDH (mouse; Santa Cruz Biotechnology, Dallas, TX, USA, sc-32233, 1 : 1000), integrin b1 (rabbit; Abcam, ab183666, 1 : 1200), b-Actin (mouse; Sigma-Aldrich, MO, USA, A5441, 1 : 1000), Paxillin (rabbit; Abcam, ab32084, 1 : 1000), pFAK (Y397) (rabbit; CST, 3283S, 1 : 1500), and FAK (mouse; BD, 610088, 1 : 1000).

Techniques: Migration, Control, Plasmid Preparation, Knockdown, Mutagenesis

Journal: The FEBS journal

Article Title: Cadherin-6 controls neuronal migration during mouse neocortical development via an integrin-mediated pathway.

doi: 10.1111/febs.70150

Figure Lengend Snippet: Fig. 4. CDH6 regulates neuronal migration in the developing cortex in an RGD motif-dependent manner. (A) E14.0 embryos were electroporated with Ta1 empty vector or Ta1-Cdh6 RGD mut* with Cdh6 KD vector and CAG-EGFP, and the distribution of EGFP- labeled cells was examined at E18.0. (B) Graphs show the quantification of cell migration in (A) (Ta1 empty, n = 6; Ta1-Cdh6 RGD mut*, n = 5). Data are mean SEM. No significant difference was observed (Student’s t-test). Sections in (A) were stained with DAPI (magenta). Scale bar: 50 lm in (A). KD, knockdown; mut, mutant.

Article Snippet: The following primary antibodies were used in this study: CDH6 (sheep; R&D Systems, AF2715, 1 : 200), HA (rat; Roche, 11867423001, 1 : 1500), GAPDH (mouse; Santa Cruz Biotechnology, Dallas, TX, USA, sc-32233, 1 : 1000), integrin b1 (rabbit; Abcam, ab183666, 1 : 1200), b-Actin (mouse; Sigma-Aldrich, MO, USA, A5441, 1 : 1000), Paxillin (rabbit; Abcam, ab32084, 1 : 1000), pFAK (Y397) (rabbit; CST, 3283S, 1 : 1500), and FAK (mouse; BD, 610088, 1 : 1000).

Techniques: Migration, Plasmid Preparation, Labeling, Staining, Knockdown, Mutagenesis

Journal: The FEBS journal

Article Title: Cadherin-6 controls neuronal migration during mouse neocortical development via an integrin-mediated pathway.

doi: 10.1111/febs.70150

Figure Lengend Snippet: Fig. 6. CDH6 regulates neuronal migration in the developing cortex through activation of integrin b1. (A) In vitro integrin activation assay. Control or Cdh6 KD vectors together with CAG-EGFP were introduced into E14.0 cortex by IUE. Three days after IUE, GFP-labeled cells were collected by FACS, and then plated on PLL-coated dishes. The amount of activated integrin b1 was quantified by the amount of bound 9EG7 antibody. (B, C) Normalized graphs of total integrin b1/b-Actin ratio (B) and activated integrin b1/b-Actin ratio (C), respectively (n = 4). Each bar represents the mean relative intensity SEM of the signal. *P = 0.048 (Student’s t-test). (D) E14.0 embryos were electroporated with Ta1 empty vector or Ta1-integrin b1 with Cdh6 KD and CAG-EGFP, and the distribution of EGFP-labeled cells was examined at E18.0. (E) Graphs show the quantification of cell migration in (D) (Ta1 empty vector, n = 6; Ta1-integrin b1, n = 7). Statistically significant differences were observed in bin 10 (**P = 0.006) (Student’s t-test). Data are mean SEM. (F) Immunostaining with 9EG7 (red) and for EGFP (green) using sections from the E18.0 cerebral cortex, which were electroporated with Ta1 empty vector (left) or Ta1-integrin b1 (right) with Cdh6 KD and CAG-EGFP vectors at E14.0. 9EG7 signal was detected in the EGFP-positive neurons of Ta1-integrin b1- electroporated samples (arrows in right panels), but not in those of Ta1 empty vector-electroporated samples (arrows in left panels) (n = 4). (G) High magnification images of EGFP-positive neurons pointed by arrows in (F) (n = 4). (H) Percentage of 9EG7-positive cells out of total GFP cells in the superficial and deep halves of the cortical wall of E18.0 cerebral cortex, which were electroporated with Ta1-integrin b1 with Cdh6 KD and CAG-EGFP vectors at E14.0. Data are mean SEM. Statistical analyses were performed using Student’s t-test (**P = 0.014, n = 3). Sections in (D) were stained with DAPI (magenta). Scale bars: 50 lm in (D, F); 10 lm in (G). FACS, fluorescence activated cell sorting; IUE, in utero electroporation; KD, knockdown; PLL, poly-L-Lysine.

Article Snippet: The following primary antibodies were used in this study: CDH6 (sheep; R&D Systems, AF2715, 1 : 200), HA (rat; Roche, 11867423001, 1 : 1500), GAPDH (mouse; Santa Cruz Biotechnology, Dallas, TX, USA, sc-32233, 1 : 1000), integrin b1 (rabbit; Abcam, ab183666, 1 : 1200), b-Actin (mouse; Sigma-Aldrich, MO, USA, A5441, 1 : 1000), Paxillin (rabbit; Abcam, ab32084, 1 : 1000), pFAK (Y397) (rabbit; CST, 3283S, 1 : 1500), and FAK (mouse; BD, 610088, 1 : 1000).

Techniques: Migration, Activation Assay, In Vitro, Control, Labeling, Plasmid Preparation, Immunostaining, Staining, FACS, In Utero, Electroporation, Knockdown

Journal: The FEBS journal

Article Title: Cadherin-6 controls neuronal migration during mouse neocortical development via an integrin-mediated pathway.

doi: 10.1111/febs.70150

Figure Lengend Snippet: Fig. 7. KD and overexpression of Cdh6 did not alter the levels of Paxillin protein and phosphorylated AKT. (A) Immunostaining for Paxillin (top, red) or pFAK (red, bottom) and EGFP (green) using sections from the E18.0 cerebral cortex, which were electroporated with control (n = 3) or Cdh6 KD vector (n = 3) (left) or control (CAG empty) (n = 3) or CAG-Cdh6-HA vectors (n = 3) (right) with CAG-EGFP vectors at E14.0. (B) Western blot analysis of neocortical cells electroporated with CAG empty or CAG-Cdh6- HA vectors using antibodies against HA, Paxillin, pFAK, FAK, and GAPDH (a loading control) (n = 3). Sections in (A) were stained with DAPI (magenta). Scale bar: 50 lm in (A). KD, knockdown.

Article Snippet: The following primary antibodies were used in this study: CDH6 (sheep; R&D Systems, AF2715, 1 : 200), HA (rat; Roche, 11867423001, 1 : 1500), GAPDH (mouse; Santa Cruz Biotechnology, Dallas, TX, USA, sc-32233, 1 : 1000), integrin b1 (rabbit; Abcam, ab183666, 1 : 1200), b-Actin (mouse; Sigma-Aldrich, MO, USA, A5441, 1 : 1000), Paxillin (rabbit; Abcam, ab32084, 1 : 1000), pFAK (Y397) (rabbit; CST, 3283S, 1 : 1500), and FAK (mouse; BD, 610088, 1 : 1000).

Techniques: Over Expression, Immunostaining, Control, Plasmid Preparation, Western Blot, Staining, Knockdown