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