Journal: ACS chemical neuroscience

Article Title: Chronic Social Isolation Stress during Peri-Adolescence Alters Presynaptic Dopamine Terminal Dynamics via Augmentation in Accumbal Dopamine Availability

doi: 10.1021/acschemneuro.8b00360

Figure Lengend Snippet: Assessment of several terminal protein expression levels. Relative protein expression levels of (A) VMAT2 (n = 7 in both groups), (B) Synaptigyrin-3 (aGH: n = 6; aSI: n = 5), (C) Syntaxin-1 (aGH: n = 7; aSI: n = 6), and (D) Munc13-3 (aGH: n = 8; aSI: n = 8) were measured. None of these proteins had different expression levels in aGH and aSI rats. (insets) The representative Western blot images, with the respective protein and actin for comparison. Group housed, aGH, blue; Socially isolated, aSI, red.

Article Snippet: Subsequently, blots were incubated with agitation for 2 h at room temperature in TBS-T/5% bovine serum albumin (05470; Sigma-Aldrich) solution containing the following primary antibody concentrations: VAMT2 (1:2000; AB1598P; Millipore Sigma); Synaptogyrin-3 (1:1000; ab106460; abcam); Syntaxin-1 (1:1000; ANR-002; Alomone laboratories).

Techniques: Expressing, Western Blot, Isolation

Journal: CNS Neuroscience & Therapeutics

Article Title: DL ‐3‐n‐Butylphthalide Protects Mitochondria Against Ischemia/Hypoxia Damage via Suppressing GCN5L1 ‐Mediated Drp1 Acetylation in Neurons and Mouse Brains

doi: 10.1002/cns.70682

Figure Lengend Snippet: NBP reversed the upregulation of Drp1 and GCN5L1 expression by ischemia/hypoxia. (A) Neuro‐2a cells were exposed to OGD and treated or not with NBP for 4 h, and then cellular lysates were prepared and analyzed by Western blot for Drp1 and GCN5L1. The representative blots are shown on the left, whereas band intensities that were measured and normalized to β‐Actin are shown on the right. Data are represented as mean ± SD, * p < 0.05, ** p < 0.01 versus 0 h; # p < 0.05, ## p < 0.01 versus Con. n = 3. (B) Neuro‐2a cells were treated as in (A), and then immunohistochemical staining detected Drp1 and GCN5L1 expression. The representative images are shown on the left. Scale bars = 100 μm. The right panel shows quantitative analysis of immunohistochemical staining. *** p < 0.001 versus 0 h; ## p < 0.01, ### p < 0.001 versus Con. n = 3. (C, D) Neuro‐2a cells were treated as in (A), and then immunofluorescent staining detected the expression of GCN5L1 and Drp1. The nucleus was stained with DAPI. The representative fluorescence images are shown on the left. Scale bars = 50 μm. The right panels show quantitative analysis of their fluorescence intensity. *** p < 0.001 versus 0 h, ### p < 0.001 versus Con. n = 3. (E) The dMCAO mice were treated or not with NBP for 3 days, and then protein extracts of brain tissues were prepared and analyzed by Western blot for Drp1 and GCN5L1. β‐Actin was included as a loading control. The representative blots are shown on the left, whereas band intensities that were measured and normalized to β‐Actin are shown on the right. Data are represented as mean ± SD, *** p < 0.001 versus Sham; # p < 0.05, and ### p < 0.001 versus Con. n = 3. (F) The dMCAO mice were treated as in (E), and immunohistochemical staining detected the expression of GCN5L1 and Drp1 on brain sections. Scale bars = 100 μm. The right panel shows quantitative analysis of immunohistochemical staining. *** p < 0.001 versus Sham, ### p < 0.001 versus Con. n = 3. (G, H) The dMCAO mice were treated as in (E), and then immunofluorescent staining detected the expression of GCN5L1 and Drp1. The nucleus was stained with DAPI. The representative fluorescence images are shown on the left. Scale bars = 100 μm. The right panels show quantitative analysis of their fluorescence intensity. *** p < 0.001 versus Sham, ### p < 0.001 versus Con. n = 3.

Article Snippet: After blocking with goat serum for 30 min, the cells were incubated overnight at 4°C with primary antibodies against Drp1 (1:200, Abcam, ab9787), GCN5L1 (1:50, Proteintech), Bax (1:200, Proteintech), Caspase‐3 (1:50, Proteintech), Bcl‐2 (1:50, Proteintech), and p‐ERK1/2 (1:50, Proteintech).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Fluorescence, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: DL ‐3‐n‐Butylphthalide Protects Mitochondria Against Ischemia/Hypoxia Damage via Suppressing GCN5L1 ‐Mediated Drp1 Acetylation in Neurons and Mouse Brains

doi: 10.1002/cns.70682

Figure Lengend Snippet: NBP decreased the acetylation of Drp1 by GCN5L1 by suppressing Drp1 interaction with GCN5L1. (A) Neuro‐2a cells were exposed to OGD and treated or not with NBP for 4 h, and then cellular lysates were prepared and immunoprecipitated with an anti‐GCN5L1 antibody. The immunoprecipitates were separated by SDS‐PAGE and immunoblotted with anti‐Drp1 antibody. IgG was used as a negative control for immunoprecipitation. (B) Neuro‐2a cells were treated as in (A), and then double immunofluorescence staining detected the expression and co‐localization of GCN5L1 (green) with Drp1 (red). The nucleus was stained with DAPI (blue). Yellow staining indicates co‐localization of GCN5L1 with Drp1. The representative fluorescence images are shown on the left. Scale bars = 50 μm and 10 μm, respectively. The right panels show immunofluorescence analysis of the colocalization of GCN5L1 with Drp1. (C) The dMCAO mice were treated or not with NBP for 3 days, protein extracts of brain tissues were prepared and immunoprecipitated with an anti‐GCN5L1 antibody. The immunoprecipitates were separated by SDS‐PAGE and immunoblotted with anti‐Drp1 antibody. IgG was used as a negative control for immunoprecipitation. (D) The dMCAO mice were treated as in (C), and double immunofluorescence staining detected the expression and co‐localization of GCN5L1 (green) with Drp1 (red) on brain sections. The nucleus was stained with DAPI (blue). Yellow staining indicates co‐localization of GCN5L1 with Drp1. The representative fluorescence images are shown on the left. Scale bars = 100 μm. The right panels show immunofluorescence analysis of the colocalization of GCN5L1 with Drp1. (E) Neuro‐2a cells were treated as in (A), and then cellular lysates were prepared and immunoprecipitated with anti‐acetyl‐lysine antibody. The immunoprecipitates were separated by SDS‐PAGE and immunoblotted with anti‐Drp1 antibody. IgG was used as a negative control for immunoprecipitation. (F) The dMCAO mice were treated as in (C), protein extracts of brain tissues were prepared and immunoprecipitated with anti‐acetyl‐lysine antibody. The immunoprecipitates were separated by SDS‐PAGE and immunoblotted with anti‐Drp1 antibody. IgG was used as a negative control for immunoprecipitation.

Article Snippet: After blocking with goat serum for 30 min, the cells were incubated overnight at 4°C with primary antibodies against Drp1 (1:200, Abcam, ab9787), GCN5L1 (1:50, Proteintech), Bax (1:200, Proteintech), Caspase‐3 (1:50, Proteintech), Bcl‐2 (1:50, Proteintech), and p‐ERK1/2 (1:50, Proteintech).

Techniques: Immunoprecipitation, SDS Page, Negative Control, Double Immunofluorescence Staining, Expressing, Staining, Fluorescence, Immunofluorescence