Journal: Science Advances

Article Title: A causal coding variant regulating alternative splicing of DOC2A at 16p.11.2 GWAS locus influences susceptibility to schizophrenia

doi: 10.1126/sciadv.adw7667

Figure Lengend Snippet: ( A ) Representative traces of mEPSC in primary hippocampal neuron infected with control (empty vector), DOC2A Full-Length , or DOC2A ∆Val217-Pro218 AAV. ( B and C ) Quantification of mEPSC amplitude and frequency. N ≥ 29 neurons. ( D ) Representative traces of mIPSC in in primary hippocampal neuron infected with control, DOC2A Full-Length , or DOC2A ∆Val217-Pro218 AAV. ( E and F ) Quantification of mIPSC amplitude and frequency. N ≥ 20 neurons. ( G ) Representative gels of Western blot. ( H to K ) Quantification of synaptophysin, VAMP-2, GAD65, and GAD67 expression in hippocampus tissue overexpressing control, DOC2A Full-Length , or DOC2A ∆Val217-Pro218 . N = 3 in each group. Full Western blotting images are shown in fig. S16. Data are presented as the means ± SEM; * P < 0.05, ** P < 0.01, and *** P < 0.001. Error bars indicate the SEM.

Article Snippet: The primary antibodies were as follows: FLAG (Cell Signaling Technology, no. 8146S; 1:1000) and DOC2A (Invitrogen, no. PA5-31345; 1:1000), GAD67 (Proteintech, no. 10408-1-AP; 1:5000), GAD65 (Cell Signaling Technology, no. 5843T; 1:1000), synaptophysin (Sigma-Aldrich, no. S5768; 1:500), VAMP-2 (Synaptic Systems, no. 104211; 1:2000), α-tubulin (Proteintech, no. 66031-1-Ig; 1:20,000), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Proteintech, no. 10494-1-AP; 1:20,000).

Techniques: Infection, Control, Plasmid Preparation, Western Blot, Expressing

Journal: Nature neuroscience

Article Title: A tau homeostasis signature is linked with the cellular and regional vulnerability of excitatory neurons to tau pathology

doi: 10.1038/s41593-018-0298-7

Figure Lengend Snippet: ( a , b) Representative sm-FISH images of the co-staining of EX neuronal marker ( SLC17A7 , red), IN neuronal marker ( GAD1 , purple), and target probe ( MAPT , MAPK1 , FKBP5 and ENC1 , green) in the EC ( a ) and the BA9 ( b ) of human brain without pathological hallmarks of neurodegenerative diseases (54–66 years old); dotted ovals represent individual EX or IN neurons. Three independent experiments were repeated with similar results. Scale bar, 10 μm. ( c , d ) Comparison of the number of single RNAs of the target probe in individual EX and IN neuron in the EC ( c ) and the BA9 ( d ) regions (n = 4 human brains, 10 neurons from each case). Data are presented as mean ± SEM. The statistical significance was assessed by two-tailed unpaired t test with Welch’s correction. *** P < 0.0001 vs IN neurons (The statistic is t=8.061, df=47; t=6.181 df=42; t=10.77, df=39 (c); and t=7.981, df=48; t=4.675, df=56; t=12.60, df=41 (d), respectively.).

Article Snippet: Rat anti-somatostatin (SST) (Cat# MAB354) and mouse anti-NeuN (Cat# MAB377) monoclonal antibody and goat anti-GAD1 (Cat# AF2086) polyclonal antibody were purchased from Millipore and R&D Systems, respectively.

Techniques: Staining, Marker, Comparison, Two Tailed Test

Journal: Nature neuroscience

Article Title: A tau homeostasis signature is linked with the cellular and regional vulnerability of excitatory neurons to tau pathology

doi: 10.1038/s41593-018-0298-7

Figure Lengend Snippet: ( a) Representative western blot images of primary cortical neurons transduced with lentivirus expressing scrambled BAG3 or shBAG3, or overexpressing BAG3 (OE) as described in Online Methods. GAPDH is a housekeeping protein used as the loading control. Three independent experiments were repeated with similar results. Full length of the blot can be found in the . ( b ) The percentage of EX and IN neurons (n = 55 from 11 coverslips each group) with 12E8 (pS262 and/or pS356 tau)-positive (+) puncta (≥ 5) in the neurites was quantified as described in Online Methods. ( c ) Representative immunocytochemical images of 12E8+ (red) puncta (white arrow heads) in the neurites of TBR1+ (green) EX neurons. White arrow indicates a neuron with high expression of TBR1; yellow arrow indicates a neuron with low expression of TBR1. GAD1+ (purple) IN neurons were also transduced with shBAG3 lentivirus and tau was shown to accumulate in neurites (white arrowheads). Three independent experiments were repeated with similar results. ( d ) Representative immunocytochemical images of tau inclusions (green) in TBR1+ (red) EX neurons (white dotted circle) (white arrow, high expression of TBR1; yellow arrow, low expression of TBR1) and tau inclusions (green) in GAD1+ (purple) IN neurons (yellow dotted circle) transduced with different lentiviruses as described in Online Methods. The nuclei were counterstained with Hoechst33342 (blue). Three independent experiments were repeated with similar results. Scale bars, 50 μm ( c ); 20 μm ( d ). ( e , f ) The quantitation of the number of TBR1+ EX and GAD1+ IN neurons with tau inclusions (n = 80 region of interests (ROI) from 4 coverslips per group). ( b , e and f ) Data are presented as mean ± SEM. Statistical significance was assessed by nonparametric Kruskal-Wallis test with the post hoc test of Dunn’s multiple comparisons. *** P < 0.0001 vs neurons transduced with scramble BAG3 (The Kruskal-Wallis statistic is 34.54, 164.6 and 20.09, respectively.).

Article Snippet: Rat anti-somatostatin (SST) (Cat# MAB354) and mouse anti-NeuN (Cat# MAB377) monoclonal antibody and goat anti-GAD1 (Cat# AF2086) polyclonal antibody were purchased from Millipore and R&D Systems, respectively.

Techniques: Western Blot, Transduction, Expressing, Control, Quantitation Assay