Journal: Scientific Reports

Article Title: Disrupted-in-schizophrenia-1 (DISC1) Regulates Endoplasmic Reticulum Calcium Dynamics

doi: 10.1038/srep08694

Figure Lengend Snippet: (a) The subcellular fractionation procedure using sucrose gradients ( 1 ) and subcellular localization of DISC1 ( 2i ) or DISC1 fragments ( 3i ) in HEK293 cells. Calnexin, mitofilin and α-tubulin were used as ER, mitochondrial and cytosolic markers, respectively. (b) Co-localization between Flag-DISC1 and GFP-ER in HEK293 cells ( i ), Flag-mDISC1 and GFP-ER in mouse hippocampal neurons at DIV10 ( ii ). Scale bars represent 10 μm. (c) Electron micrograph with pre-embedding immunogold staining. Ultrastructure of Flag-DISC1 in HEK293 cells showing the existence of DISC1 in the ER (red arrowhead) and mitochondria (black arrowheads) ( i ). Double immunogold labeling of Flag-DISC1 with calnexin in HEK293 cells ( ii ) and endogenous mDISC1 with calnexin in adult mouse brain sections ( iii ). Immunogold particles (6 nm) for Flag-DISC1 and endogenous mDISC1 are indicated with black arrows and 15 nm immunogold particles for calnexin with red arrows. The magnified images are represented with bold lines and scale bars represent 200 nm ( i ) and 100 nm or 50 nm ( ii , iii ). Western blotting was performed under the same experimental conditions in each panel and full-length blots are presented in .

Article Snippet: Goat anti-mouse DISC1 (N16, SC-47990), goat and rabbit anti-IP3R1 (SC-6093, SC-28614) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Fractionation, Staining, Labeling, Western Blot

Journal: Scientific Reports

Article Title: Disrupted-in-schizophrenia-1 (DISC1) Regulates Endoplasmic Reticulum Calcium Dynamics

doi: 10.1038/srep08694

Figure Lengend Snippet: (a) Co-immunoprecipitation of Flag-DISC1 with GFP-EXOC1 in HEK293 cells. (b) Co-localization of Flag-mDISC1 and endogenous mEXOC1 in the ER of mouse hippocampal neurons at DIV10. Scale bars represent 10 μm. (c) Co-immunoprecipitation of Flag-DISC1 with GFP-EXOC1 in ER and cytosolic fractions of HEK293 cells. 400 μg of ER and 1 mg cytosolic protein preparations were used. (d) Trypsin sensitivity of Flag-DISC1 and GFP-EXOC1 in the ER fraction of HEK293 cells. GFP-ER (ER luminal construct), Flag-DISC1 and GFP-EXOC1 in intact or permeabilized aliquots of the ER fraction were degraded by trypsin in a dose-dependent manner. Band intensities were quantified with Image J and normalized to the control lane (no trypsin treatment) (n = 3). Error bars represent mean ± SEM. **; P < 0.01 (two-way ANOVA). Western blotting was performed under the same experimental conditions in each panel and full-length blots are presented in .

Article Snippet: Goat anti-mouse DISC1 (N16, SC-47990), goat and rabbit anti-IP3R1 (SC-6093, SC-28614) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Construct, Control, Western Blot

Journal: Scientific Reports

Article Title: Disrupted-in-schizophrenia-1 (DISC1) Regulates Endoplasmic Reticulum Calcium Dynamics

doi: 10.1038/srep08694

Figure Lengend Snippet: (a) Enhanced ER localization of Flag-DISC1 upon EXOC1 overexpression in HEK293 cells. Statistical quantification of ER localization of Flag-DISC1 was performed with densitometric analysis using Image J. DISC1 level was normalized to calnexin (n = 3). (b) Reduced ER localization of Flag-DISC1 upon EXOC1 knockdown in HEK293 cells. Statistical quantification of ER localization of Flag-DISC1 was performed with densitometric analysis using Image J. DISC1 level was normalized to calnexin (n = 3). (c) Co-immunoprecipitation of Flag-DISC1 with endogenous IP3R1 in HEK293 cells. (d) Co-immunoprecipitation of three DISC1 fragments (residues 1–350, 345–591, 586–854) with endogenous IP3R1. Statistical quantification of the IP3R1 binding levels of DISC1 was carried out via densitometric analysis using Image J (n = 3). (e) Reduced IP3R1 interaction with DISC1 upon EXOC1 knockdown in HEK293 cells. Statistical quantification of the IP3R1 binding levels of DISC1 was carried out via densitometric analysis using Image J. DISC1 level in IP lane was normalized to input lane (n = 6). (f) Reduced PKA-mediated phosphorylation of IP3R1 in DISC1 knockdown HEK293 cells ( i ) and mouse neuroblastoma CAD cells ( ii ) (n = 6 for i , 7 for ii ). Error bars represent means ± SEM. *; P < 0.05, **; P < 0.01, ***; P < 0.001 (two-tailed t -test). Western blotting was performed under the same experimental conditions in each panel and full-length blots are presented in .

Article Snippet: Goat anti-mouse DISC1 (N16, SC-47990), goat and rabbit anti-IP3R1 (SC-6093, SC-28614) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Knockdown, Immunoprecipitation, Binding Assay, Phospho-proteomics, Two Tailed Test, Western Blot

Journal: Scientific Reports

Article Title: Disrupted-in-schizophrenia-1 (DISC1) Regulates Endoplasmic Reticulum Calcium Dynamics

doi: 10.1038/srep08694

Figure Lengend Snippet: (a) Enhanced IP3R-dependent ER calcium dynamics upon DISC1 or EXOC1 knockdown in HEK293 cells. Calcium response graph under 50 μM ATP stimulation ( i ), statistically analyzed average area ( ii ) and amplitudes ( iii ) of the graph (n = 30 for control shRNA, 29 for EXOC1 shRNA, 42 for DISC1 shRNA, 21 for DISC1 shRNA + EXOC1 shRNA). (b) Reduced IP3R-dependent ER calcium dynamics in HEK293 cells overexpressing DISC1 or EXOC1 under 50 μM ATP stimulation. Based on ER calcium response curves ( i ), statistically analyzed average area ( ii ) and amplitude ( iii ) were shown (n = 24 for GFP vector, 21 for GFP-EXOC1, 17 for GFP-DISC1, 35 for GFP-DISC1 Δ 1-351, 18 for GFP-DISC1+GFP-EXOC1). (c) ER calcium response curves under 50 nM bradykinin stimulation in mDISC1 and mEXOC1 knockdown neuroblastoma CAD (Cath.-a-differentiated) cells ( i ). Area and amplitude of ER calcium response curve under 50 nM BK (bradykinin) were analyzed ( ii ) (n = 41 for control shRNA, 26 for mEXOC1 shRNA, 39 for mDISC1 shRNA). (d) Enhanced IP3R-dependent ER calcium dynamics upon mDISC1 or mEXOC1 knockdown in DIV7 mouse hippocampal neurons. Representative live images of calcium response to ATP stimulation in control, mEXOC1, and mDISC1 knockdown conditions ( i ), calcium response graph under 10 mM ATP stimulation ( ii ), statistically analyzed average area ( iii ) and amplitudes ( iv ) of the graph (n = 17 for control shRNA, 13 for mEXOC1 shRNA, 11 for mDISC1 shRNA). Error bars represent means ± SEM. *; P < 0.05, **; P < 0.01, ***; P < 0.001 (two-tailed t -test).

Article Snippet: Goat anti-mouse DISC1 (N16, SC-47990), goat and rabbit anti-IP3R1 (SC-6093, SC-28614) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Control, shRNA, Plasmid Preparation, Two Tailed Test

Journal: Scientific Reports

Article Title: Disrupted-in-schizophrenia-1 (DISC1) Regulates Endoplasmic Reticulum Calcium Dynamics

doi: 10.1038/srep08694

Figure Lengend Snippet: (a) Live calcium images of cultured hippocampal neurons of wild-type (WT) and DISC1 ( Δ 2-3) mouse embryos in response to ATP ( i ). ER calcium response graph under 10 mM ATP ( ii ), statistically analyzed area ( iii ), amplitude ( iv ) (n = 88 for WT, 74 for DISC1 ( Δ 2-3)). (b) Reversal of the exaggerated ER calcium dynamics in DISC1 ( Δ 2-3) hippocampal neurons by haloperidol (0.1 μM). From the ER calcium response curves upon 30 mM ATP stimulation ( i ), average area under curves ( ii ), amplitudes ( iii ) and drug-responsive portions ( iv ) of the curves were analyzed. (n = 31 for WT+vehicle, 28 for WT+haloperidol, 26 for DISC1 ( Δ 2-3)+vehicle, 27 for DISC1 ( Δ 2-3)+haloperidol). Error bars represent means ± SEM. *; P < 0.05, **; P < 0.01, ***; P < 0.001 (two-tailed t -test).

Article Snippet: Goat anti-mouse DISC1 (N16, SC-47990), goat and rabbit anti-IP3R1 (SC-6093, SC-28614) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Cell Culture, Two Tailed Test

Journal: Molecular Biology of the Cell

Article Title: Cdc42 negatively regulates endocytosis during apical membrane maintenance in live animals

doi: 10.1091/mbc.E18-10-0615

Figure Lengend Snippet: Cdc42-depletion results in loss of PAR6 and F-actin at the APM without altering polarity and junction permeability. (A, B) Cdc42 fl/fl-mT/mG mice were transfected with Adeno-Cre and processed for immunofluorescence as described in the legend to . Samples were labeled with iFluor 405-phalloidin (A, red) and an antibody against PAR6 (A, green) or with antibodies against AQP5 (B, red) and NKCC1 (B, green). Dotted lines outline the Cre-expressing cells. Graphs show the analysis of the levels of PAR6 (A), F-actin (A), AQP5 (B), and NKCC1 (B) in Cre-expressing (Cdc42 − ) and Cre-nonexpressing (Cdc42 + ) cells. (PAR6, N = 25 ICs, 11 acinar cells from three animals; F-actin, N = 17 APM and BLM, 12 acinar cells from four animals; AQP5, N = 12 APM and BLM, 9 acinar cells from three animals; and NKCC1, N = 17 APM and BLM, 17 acinar cells from five animals). ns = not significant, paired t test. (C, D) Three kDa Cascade Blue Dextran (White) was retrodiffused into the Wharton’s duct of anesthetized Cdc42 fl/fl-mT/mG mice after 3 wk from Adeno-Cre transfection. (C) The SMGs were exposed and imaged by ISMic. Time 0 represents the point at which dextran is detected in IC. Arrow and arrowhead in time 0 shows normal IC and expanded IC, respectively (C and Supplemental Movie S2). (D) Quantification of dextran fluorescence intensity within (A, C) and outside (B, D) in Cre-negative cells (A, B) and Cre-positive cells (C, D). Graph shows a representative experiment.

Article Snippet: NKCC1 (N-16) , Goat , Santa Cruz , sc-21545 , 1:100.

Techniques: Permeability, Transfection, Immunofluorescence, Labeling, Expressing, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: Cdc42 negatively regulates endocytosis during apical membrane maintenance in live animals

doi: 10.1091/mbc.E18-10-0615

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: NKCC1 (N-16) , Goat , Santa Cruz , sc-21545 , 1:100.

Techniques: