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: Frontiers in Cellular Neuroscience

Article Title: Platelet Activating Factor Enhances Synaptic Vesicle Exocytosis Via PKC, Elevated Intracellular Calcium, and Modulation of Synapsin 1 Dynamics and Phosphorylation

doi: 10.3389/fncel.2015.00505

Figure Lengend Snippet: cPAF increases synapsin I phosphorylation and synapsin I dispersion from presynaptic boutons during stimulation. (A–D) Hippocampal neurons expressing synapsin I-GFP and Tdtomato were treated with 1 μM cPAF for 2 min then stimulated with 900 pulses at 10 Hz. (A) Synapsin I-GFP (Syn-GFP) is clustered at presynaptic boutons while tdtomato fills the entire axon. Scale bar = 2 μm. (B) Syn-GFP fluorescence at synapses decreases upon stimulation then reclusters within the bouton. Scale bar = 2 μm. (C) Timelapse measurements of the change in Syn-GFP fluorescence intensity obtained from the boutons numbered in (A) normalized to the fluorescence at time 0. (D) Average Δ F / F 0 in syn-GFP fluorescence from all boutons treated with 1 μM cPAF or vehicle. Error bars ±SEM (cPAF: 141 boutons from three coverslips; vehicle: 240 boutons from four coverslips;). Non-linear regression of decay part of synapsin I dispersion curve followed by testing the null hypothesis of one curve for all data sets has p < 0.0001. Statistical analysis comparing cPAF to vehicle treated boutons at the end of stimulation (1.5 min) was done using the Student’s t -test p = 0.0005. (E) Representative western blots showing phosphorylated synapsin I (pSynapsin) at Site 3 (Ser603) and Site 1 (Ser9) and GAPDH immunoreactivity from cell lysates obtained from primary hippocampal cultures that were treated 0, 2, or 20 min with 1 μM cPAF. Blot on right was additionally treated for a total of 30 min with 50 μM APV and 10 μM CNQX to block glutamatergic neurotransmission. (F) pSynapsin/GAPDH ratios calculated by densitometric scanning of the blots. Data are means ± SEM with n = 4 (two independent experiments each performed in duplicate). Statistical analysis: one-way ANOVA followed by Dunnett’s multiple comparison test to 0 min control. ANOVA: Site 3, p = 0.098; Site 3 with APV/CNQX, p = 0.024; Site 1, p = 0.211; Site 1 with APV/CNQX, p = 0.126. Dunnett’s multiplicity adjusted p -values (compared to 0 min control): ∗ p < 0.1 ∗∗ p < 0.05.

Article Snippet: Membranes were blocked with 5% milk in TBSt for 30 min and probed with primary antibodies (phospho-synapsin Ser9 (Cell Signaling Technologies #2311), phospho-synapsin I Ser603 (Rockland 612-401-C95), and GAPDH (EMD Millipore: Calbiochem CB1001) in 3% BSA in TBSt at 4° overnight.

Techniques: Expressing, Fluorescence, Western Blot, Blocking Assay

Journal: Respiratory Research

Article Title: Bhlhe40 deficiency attenuates LPS-induced acute lung injury through preventing macrophage pyroptosis

doi: 10.1186/s12931-024-02740-2

Figure Lengend Snippet: Bhlhe40 deficiency alleviates GSDMD-mediated pyroptosis and caspase-1 and caspase-11 pathways in LPS-induced ALI mice. ( A ) Representative Western blot of GSDMD FL and GSDMD NT in the lung tissues of mice. ( B ) Representative Western blot of cleaved IL-1β in the lung tissues of mice. ( C ) Representative images of F4/80 and GSDMD NT double-immunofluorescence staining of LPS-induced lungs. Scale bar = 50 μm and 20 μm. (D) Representative Western blot of pro-caspase-1, cleaved caspase-1, pro-caspase-11, cleaved caspase-11, NLRP3, ASC, TLR4 and MYD88 in the lung tissues of mice. n = 6

Article Snippet: Antibodies used for Western blot, immunohistochemistry and Immunofluorescence included Bhlhe40 (NB100-1800SS) from Novus (Centennial, CO, United States), GAPDH (ab181602), GSDMD (ab209845), Caspase-11 (ab180673) from Abcam (Cambridge, United Kingdom), F4/80 (70076T), NLRP3 (15101 S), ASC (67824T) from Cell Signal Technology (Danvers, MA, United States), GSDMD-NT (orb1495171) from biorbyt (Cambridge, United Kingdom), Caspase-1 (A0964) from Abclone (Wuhan, China), and TLR4 (66350-1-Ig), MYD88 (67969-1-Ig) from Proteintech (Rosemont, IL, United States).

Techniques: Western Blot, Double Immunofluorescence Staining

Journal: Respiratory Research

Article Title: Bhlhe40 deficiency attenuates LPS-induced acute lung injury through preventing macrophage pyroptosis

doi: 10.1186/s12931-024-02740-2

Figure Lengend Snippet: Bhlhe40 deficiency inhibits GSDMD-mediated pyroptosis through both canonical and non-canonical pathways in BMDMs and in RAW264.7 cell line. ( A - C ) Representative Western blot of GSDMD FL , GSDMD NT , cleaved IL-1β, pro-caspase-1, cleaved caspase-1, pro-caspase-11, cleaved caspase-11, NLRP3 and ASC in BMDMs. ( D - E ) RAW264.7 cells were transfected with three Bhlhe40 siRNAs (siRNA#1, siRNA#2 and siRNA#3) or normal control siRNA (NC) for 48 h. The protein levels of Bhlhe40 were detected by western blots and quantified analysis. ( E - F ) Representative Western blot of GSDMD FL , GSDMD NT , cleaved IL-1β, pro-caspase-1, cleaved caspase-1, pro-caspase-11, cleaved caspase-11, NLRP3 and ASC in RAW246.7 cells. n = 3. Data are shown as the mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. *p < 0.05, ns p > 0.05

Article Snippet: Antibodies used for Western blot, immunohistochemistry and Immunofluorescence included Bhlhe40 (NB100-1800SS) from Novus (Centennial, CO, United States), GAPDH (ab181602), GSDMD (ab209845), Caspase-11 (ab180673) from Abcam (Cambridge, United Kingdom), F4/80 (70076T), NLRP3 (15101 S), ASC (67824T) from Cell Signal Technology (Danvers, MA, United States), GSDMD-NT (orb1495171) from biorbyt (Cambridge, United Kingdom), Caspase-1 (A0964) from Abclone (Wuhan, China), and TLR4 (66350-1-Ig), MYD88 (67969-1-Ig) from Proteintech (Rosemont, IL, United States).

Techniques: Western Blot, Transfection, Control