Journal: iScience

Article Title: Chronic HDM exposure shows time-of-day and sex-based differences in inflammatory response associated with lung circadian clock disruption

doi: 10.1016/j.isci.2023.107580

Figure Lengend Snippet: Serum levels of total and HDM-specific immunoglobulins show a time-of-day response and sex-based differences to chronic HDM exposure Total IgE, Total IgG, HDM-specific IgE, HDM-specific IgG, HDM-specific IgG1, HDM-specific IgG2b, HDM-specific IgM, and HDM-specific IgA in the serum of chronic PBS- and HDM-exposed females and males at ZT0 and ZT12 were determined by ELISA. Data were expressed as ng/ml and mg/ml for total IgE and total IgG, respectively. HDM-specific immunoglobulins (IgE, IgG, IgG1, IgG2b, IgM, and IgA) in the serum were determined by commercially available ELISA kits (Chondrex, Inc.). Unable to detect HDM-specific IgG2a and IgG3 levels in PBS- and HDM-exposed mice. Data for the HDM-specific immunoglobulins were expressed as absorbance at 450 nm or μg/ml. Data are shown as mean ± SEM, Two-way ANOVA followed by Tukey’s multiple comparison test (n = 5/group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, compared to respective control (PBS) at ZT0 or ZT12; # p < 0.05, compared to HDM at ZT0 vs. ZT12; # # p < 0.01, compared to HDM at ZT0 vs. ZT12. Summary statistics for interaction between Treatment x Sex x Time were analyzed using generalized linear modeling using R (see Table S2 ).

Article Snippet: Mouse Anti-House Dust Mite (HDM) IgG2b Antibody Assay Kit , Chondrex, Inc. , Cat# 3035.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: iScience

Article Title: Chronic HDM exposure shows time-of-day and sex-based differences in inflammatory response associated with lung circadian clock disruption

doi: 10.1016/j.isci.2023.107580

Figure Lengend Snippet:

Article Snippet: Mouse Anti-House Dust Mite (HDM) IgG2b Antibody Assay Kit , Chondrex, Inc. , Cat# 3035.

Techniques: Purification, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Software

Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Schematic diagram showing domain organization and variant fragments of Doc2B and Munc13-1. ( B ) Binding of MUN to GST-Doc2B FL or its variant fragments measured by GST pull-down experiments and quantification of the binding. ( C ) ITC-based measurements of MUN binding to GST-Doc2B 13 to 37 (Mid, left) and to GST-Doc2B 13 to 80 (Mid-L, right). ( D ) ITC-based measurements of the binding affinities between GST-Doc2B FL or its variant fragments and MUN. ( E ) 2D 1 H- 15 N HSQC spectra of 13 C/ 15 N-labeled Doc2B 1 to 80 before (black) and after (red) addition of MUN. Cross-peaks of residues that are chosen for mutation to detect MUN binding are labeled along with their corresponding residue number (cyan). ( F ) Peak intensity alteration of 13 C/ 15 N-labeled Doc2B 1 to 80 protein expressed as ratio between integrated peak volumes after ( V ) and before ( V 0 ) addition of unlabeled MUN. ( G ) Binding of Doc2B FL or its variant mutations to GST-MUN measured by GST pull-down experiments and quantification of the binding. ( H ) Binding of MUN or its variant fragments to GST-Doc2B FL measured by GST pull-down experiments and quantification of the binding. Red asterisk shows the band of bound MUN-BC. ( I ) Binding of MUN or its variant mutations to GST-Doc2B FL measured by GST pull-down experiments and quantification of the binding. Data of GST pull-down experiments are processed by ImageJ (National Institutes of Health) and presented as means ± SEM ( n = 3). Statistical significance and P values were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. * P < 0.05; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Rabbit polyclonal pY36 antibody, which recognizes residues 32 to 40 of Doc2B, was generated by ABclonal.

Techniques: Variant Assay, Binding Assay, Labeling, Mutagenesis, Residue

Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Illustration of the FRET assay for detecting MUN-catalyzed SNARE complex assembly starting from the Munc18-1/Syx1 (1 to 261) complex in the presence of Syb2 (29 to 96), SN25, and MUN. FRET signal between BDPY-labeled Syb2 S61C (donor) and TMR-labeled SN25 S187C (acceptor) was monitored. ( B to D ) MUN-catalyzed SNARE complex assembly by addition of Doc2B Mid, Doc2B Mid-L, or Doc2B FL, respectively (B), addition of different concentrations of Doc2B Mid (C), and addition of MUN NFAA or Doc2B I20A, which disrupts Doc2B-MUN interaction (D). Decrease of donor fluorescence at 1500 s is shown in the column at the right of the chart. ( E ) Illustration of Munc13-catalyzed lipid mixing between liposomes bearing Syb2 (1 to 116) and liposomes bearing the Munc18-1/Syx1 (1 to 288) complex in the presence of SN25, Syt1 C2AB, Ca 2+ , and Munc13-1 (C 1 C 2 BMUN). Donor (NBD) fluorescence was monitored at 538 nm. ( F to H ) Munc13-catalyzed lipid mixing by addition of Doc2B Mid, Doc2B Mid-L, or Doc2B FL, respectively (F); addition of C 1 C 2 BMUN NFAA or Doc2B I20A (G). Munc13-catalyzed lipid mixing at 1000 s is shown in (H). F 1 , fluorescence intensity observed as a function time; F 0 , initial fluorescence intensity. Data are presented as means ± SEM ( n = 3). Statistical significance and P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: Rabbit polyclonal pY36 antibody, which recognizes residues 32 to 40 of Doc2B, was generated by ABclonal.

Techniques: Labeling, Fluorescence, Liposomes

Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Immunostaining against Doc2B (green) and EphB2 (red) in the mouse hippocampus region. Nuclear DNA was labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 500 μm. ( B ) Separation of pre- and postsynaptic densities from purified synaptosomes. Syn, synaptosome; Pre, presynaptic elements; Post, postsynaptic elements; Extra, extrajunctional synaptic elements. ( C and D ) Western blot analysis of Doc2B-EphB2 interaction following cotransfection of mCherry-tagged Doc2B and Flag-tagged EphB2 in HEK293 cells. Cell lysates were immunoprecipitated by anti-mCherry antibody (C) or anti-Flag antibody (D), followed by immunoblotting with indicated antibody. ( E ) Schematic representation of EphB2 constructs. ( F and G ) Western blot analysis of Doc2B-EphB2 interaction following cotransfection of various combinations of plasmids containing EphB2 FL, EphB2-∆SP, EphB2-∆KSP, and/or mCherry-tagged Doc2B (F), EphB2 WT, KR, and/or mCherry-tagged Doc2B (G) in HEK293 cells. ( H and I ) In vitro phosphorylation assay using sumo-tagged EphB2 and GST-tagged Doc2B fragments (H) and GST-tagged Doc2B Mid or Y36F (I) in the presence or absence of ATP and APase. Asterisks show bands of GST-Doc2B Mid, Mid-L, and FL, respectively. CBB, Coomassie Brilliant Blue staining. ( J and K ) Western blot analysis of Doc2B phosphorylation following cotransfection of mCherry-tagged Doc2B WT or Y36F and/or EphB2 (J) and EphB2 WT, KR, or YYEE and/or mCherry-tagged Doc2B (K) in HEK293 cells. ( L ) Detection of Doc2B phosphorylation in cultured mouse cortex neurons by application of Fc (control) or preclustered Ephrin-B3-Fc. ( M and N ) Western blot analysis of Doc2B phosphorylation in EphB2 +/+ or EphB2 −/− mouse brains (M) in Doc2A-deficient neurons infected with Doc2B KD or control virus (N). Tubulin used as the reference protein. Data are presented as means ± SEM ( n = 3). Statistical significance and P values were determined by Student’s t test (** P < 0.01; **** P < 0.0001).

Article Snippet: Rabbit polyclonal pY36 antibody, which recognizes residues 32 to 40 of Doc2B, was generated by ABclonal.

Techniques: Immunostaining, Labeling, Purification, Western Blot, Cotransfection, Immunoprecipitation, Construct, In Vitro, Phospho-proteomics, Staining, Cell Culture, Control, Infection, Virus

Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Binding of Doc2B WT or its phosphomimetic/unphosphorylatable mutations to GST-MUN measured by GST pull-down experiments and quantification of the binding. ( B ) Binding of the MUN domain to GST-Doc2B fragments and EphB2 mixture measured by GST pull-down experiments in the presence or absence of ATP and quantification of the binding. ( C and D ) MUN-catalyzed SNARE complex assembly by addition of Doc2B WT, Y36D, or Y36F, respectively (C), and addition of Doc2B WT and EphB2 mixture in the presence or absence of ATP (D). Decrease of donor fluorescence at 1500 s is shown in the column at the right of the chart. ( E and F ) Munc13-catalyzed lipid mixing by addition of Doc2B WT, Y36D, or Y36F, respectively (E), and addition of Doc2B WT and EphB2 mixture in the presence or absence of ATP (F). Munc13-catalyzed lipid mixing at 1000 s is shown in the column at the right of the chart. F 1 , fluorescence intensity observed as a function time; F 0 , initial fluorescence intensity. Data are presented as means ± SEM ( n = 3). Statistical significance and P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Rabbit polyclonal pY36 antibody, which recognizes residues 32 to 40 of Doc2B, was generated by ABclonal.

Techniques: Binding Assay, Fluorescence

Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A and B ) Representative traces (A) and quantification of mEPSC frequency (B) in Doc2A −/− neurons (EGFP, n = 15; Doc2A WT, n = 15; Doc2B WT, n = 15). ( C and D ) Representative traces (C) and quantification of mEPSC frequency (D) in Doc2A −/− neurons expressing EGFP ( n = 16), Doc2B WT ( n = 16), Doc2B D303N ( n = 16), Doc2B (81 to 412) ( n = 16), or Doc2B I20A ( n = 16). ( E ) Representative traces of mEPSC frequency in Doc2A +/+ (top) and Doc2A −/− (bottom) neurons after stimulation with EphrinB3. ( F and G ) Summary of the changes in mEPSC frequency after stimulation with EphrinB3 in Doc2A +/+ ( n = 16) and Doc2A −/− ( n = 18) neurons (F) and in Doc2A −/− neurons expressing EGFP ( n = 16), Doc2B WT ( n = 16), or Doc2B Y36F ( n = 16) (G). Data were normalized to the average value during the control period before stimulation. ( H ) Representative traces of evoked EPSCs in synaptic augmentation at 20, 40, and 60 s in Doc2A +/+ and Doc2A −/− neurons. ( I and J ) Normalized peak amplitudes of evoked EPSCs in Doc2A +/+ ( n = 20) and Doc2A −/− ( n = 23) neurons (I) or in Doc2A −/− neurons expressing EGFP ( n = 16), Doc2B WT ( n = 16), Doc2B I20A ( n = 17), Doc2B Y36D ( n = 15), or Doc2B Y36F ( n = 15) (J). Data are presented as means ± SEM. Recorded cells are from three independent experiments. Statistical significance and P values for (B), (D), (G), and (J) were determined by one-way ANOVA with Dunnett’s multiple comparison test, and those for (F) and (I) were determined by Student’s t test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Rabbit polyclonal pY36 antibody, which recognizes residues 32 to 40 of Doc2B, was generated by ABclonal.

Techniques: Expressing, Control, Comparison