Journal: Redox Biology

Article Title: Breast cancer susceptibility protein 1 (BRCA1) rescues neurons from cerebral ischemia/reperfusion injury through NRF2-mediated antioxidant pathway

doi: 10.1016/j.redox.2018.06.012

Figure Lengend Snippet: LV-BRCA1 treatment facilitated synaptic plasticity after MCAO. (A, B) Transfection efficiency of LV-BRCA1 in hippocampus, n = 5. (C) Western blot and quantitative analysis of PSD95, CaMKII, Synapsin I and synaptophysin, n = 6. (D) Representative micrographs of DCX staining in CA1. (E) Quantification of DCX immunofluorescence density, n = 6. Scale bar = 50 µm. Data are expressed as mean ± SD; * ** P < 0.001 versus sham group; # P < 0.05, ### P < 0.001 versus MCAO group.

Article Snippet: Membranes were blocked with 3% bovine serum albumin and 5% fat-free milk at room temperature for 1.5 h. Then membranes were incubated with primary antibodies against BRCA1 (1:1000, Abcam, UK), p53 (1:1000, Abcam, UK), Bcl-2 (1:1000, Abcam, UK), Bax (1:1000, Cell Signaling technology, USA), Cleaved Caspase-3 (1:1000, Cell Signaling technology, USA), γH2A.X (1:1000, Millipore, USA), RAD51 (1:5000, Millipore, USA), Ku70/80 (1:1000, Abcam, UK), pDNA-PKcs (1:1000, Abcam, UK), DNA-PKcs (1:1000, Abcam, UK), NRF2 (1:1000, Abcam, UK), NQO1 (1:1000, Abcam, UK), HO-1 (1:200, Santa Cruz, USA), GPX4 (1:1000, Abcam, UK), PSD95 (1:1000, Abcam, UK), CaMKII (1:1000, Abcam, UK), Synapsin I (1:200, Santa Cruz, USA), Synaptophysin (1:1000, Abcam, UK), GST (1:50000, Cell Signaling technology, USA), histone H3 (1:3000, Cell Signaling technology, USA) and β-actin (1:4000, Cell Signaling technology, USA) overnight at 4 °C, followed by incubated with HRP-conjugated secondary antibody at room temperature.

Techniques: Transfection, Western Blot, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Bassoon controls synaptic vesicle pools via regulation of presynaptic phosphorylation and cAMP homeostasis

doi: 10.1101/2021.07.22.453360

Figure Lengend Snippet: (A) Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and Bsn GT neurons without treatment (black and red) and upon treatment with adenylyl cyclase activator forskolin (gray and pink) or PKA inhibitor H89 (light grey and orange). (C, D) Quantification of the RRP and TRP fraction from experiment in A. (E) Representative immunoblot with antibody against pSer9Syn1 and total Syn1 on hippocampal tissue from WT and Bsn GT mice (F) Quantification of blots from E. (G) Scheme illustrates the PKA-dependent phosphorylation of Syn1 on Ser9 that promotes recruitment of SVs to the recycling pool. Changes in this signalling confirmed in Bsn GT are depicted in red. (H) Representative images of hippocampal neurons from WT and Bsn GT mice without treatment and treated with forskolin or H89 labelled with antibodies against pSer9Syn1 and total Syn1. (I) Quantification of staining in H. In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments performed on three independently prepared culture batches in (C and D), samples prepared from individual animals in (E) or quantified independent visual fields obtained from 2 independent culture preparations in (I). The statistical significance was assessed in C, D and I using one-way ANOVA with Tukey’s post hoc test and in F using Student’s t-test as is depicted in graphs as *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 2 μm in A and 5 μm in H.

Article Snippet: The primary antibodies were used for immunocytochemistry (ICC), Western Blot (WB), and Syt1Ab uptake in the concentration indicated as follows: rabbit antibodies against Syt1 (labelled with Oyster 550; live staining: 1:70, # 105103C3, Synaptic System, Göttingen, Germany), pThr138SNAP25 (WB:1:500, # 042077, Biomol), SNAP-25 (WB: 1:1000, # 111002, Synaptic System), pSer9Syn1 (ICC:1:1000, gift from Dr. Fabio Benfenati, IIT, Genova, Italy), pSer551Syn1 (ICC: 1:1000, WB:1:1000, gift from Dr. Anna Fassio, IIT, Genova, Italy) pSer9Syn1 (WB: 1:1000, # 2311S, Cell Signaling), PKAα cat (C-20) (WB:1:500, # sc-903, Santa Cruz), pSer145PDE4B (WB 1:200, ( )), mouse antibodies against Syn1 (ICC: 1:1000, WB: 1:1000, # 106011, Synaptic System), VGLUT1 (ICC:1:1000, # MAB5502, Millipore), guinea pig antibodies against Syn1 (ICC:1:1000, # 106104, Synaptic System), VGAT (ICC:1:1000, # 131004, Synaptic System) and sheep antibody against PDE4B (WB 1:500, ( )).

Techniques: Fluorescence, Western Blot, Phospho-proteomics, Staining, Imaging

Journal: bioRxiv

Article Title: Cryo-EM structures of α-synuclein filaments from Parkinson’s disease and dementia with Lewy bodies

doi: 10.1101/2022.07.12.499706

Figure Lengend Snippet: Sections from brain regions contralateral to those used for cryo-EM structure determination were stained with monoclonal antibody Syn1 (1:1,000). (a), Cingulate cortex from PD; (b), Cingulate cortex from PDD1; (c), Cingulate cortex from PDD2; (d), Frontal cortex from DLB1; (e), Frontal cortex from DLB2; (f), Cingulate cortex from DLB3. Scale bars: a-c, f, 100 μm; d,e, 50 μm.

Article Snippet: Before blocking, membranes were fixed with 1% paraformaldehyde for 30 min. Primary antibodies were: Syn303 [a mouse monoclonal antibody that recognizes residues 1-5 of human α-synuclein ( )] (BioLegend) at 1:4,000, Syn1 [a mouse monoclonal antibody that recognizes residues 91-99 from the NAC region of human α-synuclein ( )] (BD Biosciences) at 1:4,000 and PER4 at 1:4,000.

Techniques: Cryo-EM Sample Prep, Staining

Journal: bioRxiv

Article Title: Cryo-EM structures of α-synuclein filaments from Parkinson’s disease and dementia with Lewy bodies

doi: 10.1101/2022.07.12.499706

Figure Lengend Snippet: PER4 was used at 1:50 in (a-c). (a), PD (Cingulate cortex); (b), PDD1 (Cingulate cortex); (c), DLB3 (Cingulate cortex); Syn303, Syn1 and PER4 were used at 1:4,000 in (d-f). The brain regions used for cryo-EM were also used for immunoblotting. The arrow points to the position of monomeric α-synuclein.

Article Snippet: Before blocking, membranes were fixed with 1% paraformaldehyde for 30 min. Primary antibodies were: Syn303 [a mouse monoclonal antibody that recognizes residues 1-5 of human α-synuclein ( )] (BioLegend) at 1:4,000, Syn1 [a mouse monoclonal antibody that recognizes residues 91-99 from the NAC region of human α-synuclein ( )] (BD Biosciences) at 1:4,000 and PER4 at 1:4,000.

Techniques: Cryo-EM Sample Prep, Western Blot