anti ccl25 monoclonal antibody per dose  (R&D Systems)

Journal: Nature

Article Title: Endogenous self-peptides guard immune privilege of the central nervous system

doi: 10.1038/s41586-024-08279-y

Figure Lengend Snippet: a , Dose-response curve demonstrating reactivity of an MBP-specific hybridoma clone to MBP 160-175 and MBP 166-185 peptides; readout by IL-2 ELISA of half-logarithmic serial dilutions. Plots display mean±s.e.m and represent two independent experiments. b , Schematic created with BioRender.com. Gating strategy used to define MBP-specific CD4 + T cells by tetramer staining. c-d , CD4 + T cells isolated from draining iLNs after immunization with MBP 160-175 ; sorted for tetramer positive (MBP-specific) and tetramer negative CD4 + T cells. UMAP visualization depicts 8 CD4 + T cell subclusters ( c ) with enrichment to the T regs subcluster in tetramer positive cells, indicated by red dots ( d ). e , Q-plot of TCR clones identified in tetramer negative (top) and tetramer positive (bottom) T cells colour-coded to represent the CD4 + T cell subcluster. f , Dot plot of population markers from scRNA-seq scaled by percentage of cells expressing marker genes for each CD4 + T cell cluster. g , Violin plot comparing Tgfb1 and Ctla4 expression between tetramer negative and tetramer positive samples. Data shown as mean±s.e.m (n = 8/group, unpaired two-tailed Student’s t-test). h-i , Schematic created with BioRender.com. ( h ) and bar plot of IL-2 ELISA ( i ), demonstrating the attenuation of primary MOG-specific CD4 + T cell responses by primary MBP-specific CD4 + T cells but not by tetramer negative polyclonal CD4 + T cells; this was blunted by anti-CTLA-4 or anti-TGFβ neutralizing antibodies. Data (mean±s.e.m) represent two independent experiments; two-way ANOVA performed. j , EAE assessed in mice immunized with MOG 35-55 or MOG 35-55 + MBP 160-175 ; on days 7 and 10 post-immunization, mice were treated with isotype control or anti-IL-10 neutralizing antibodies (n = 5/group). Data (mean±s.e.m) represent three independent experiments; two-way ANOVA with Dunnett’s post-hoc test used. k-m , Schematic generated with BioRender.com. ( k ), representative flow cytometry plots ( l ), and quantifications ( m ) of tetramer staining of MBP-specific or MOG-specific CD4 + T cells in the thymus of naïve 1-month-old C57BL/6J mice (n = 4/group, mean±s.e.m, unpaired two-tailed Student’s t-test).

Article Snippet: To remove peptides that non-specifically bind to Sepharose and/or immunoglobulin, the supernatant was first incubated with polyclonal mouse immunoglobulin G (IgG, Leinco Technologies; 1.5 mg antibody per sample) bound to Sepharose 4B at 4 °C for 30 min.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Isolation, Clone Assay, Expressing, Marker, Two Tailed Test, Control, Generated, Flow Cytometry

Journal: bioRxiv

Article Title: Blebs regulate phosphoinositides distribution and promote cell survival through the Septin-SH3KBP1-PI3K axis

doi: 10.1101/2024.07.09.602823

Figure Lengend Snippet: (A)Representative images of GFP-Septin6 (upper), PIK3R1-iRFP (bottom) in MDA-MB-231 cell during cell blebbing induced by low concentration Latrunculin B. Black arrows indicate Septin6 and PIK3R1 colocalization at bleb neck. Scale bar, 5 μm. (B)Representative images of GFP-Septin6 and PIK3R1-iRFP in a cell expressing SEPT2(33-306) polymerization mutant. Black arrows indicate bleb neck. (C)Surface renderings of intracellular PI(4,5)P2, PI(3,4,5)P3 mean intensity within 1Lµm of cell surface in cell expressing SEPT2(33-306) polymerization mutant. Scale bar, 5 μm. (D)Time lapse images of MDA-MB-231 cells expressing PI(4,5)P2 sensor PLCδ-PH-mMaple3 (left) or PI(3,4,5)P3 sensor Akt-PH-mMaple3 (middle) after transient photoconversion at specific membrane sites in cell expressing SEPT2(33-306) polymerization mutant. Green dashed boxes are photoconverted sites. Other colored dashed boxes marked sensor intensity are calculated in right panel. Scale bar, 5 μm. (E)Representative images and surface renderings of PI(4,5)P2 and PI(3,4,5)P3 intensity after treated with 10 µM LY294002 (PI3K inhibitor) 10 min. (F)MBP pulldown assay showing the interaction between Septin6 and Septin7 (on the left) or PIK3R1 (on the right). Samples were analyzed by SDS-PAGE and stained by Coomassie brilliant blue (N = 2 experiments, repeats are biological). (G)Co-immunoprecipitation assay showing interaction between PIK3R1-Flag and Septin6. (N = 3 experiments, repeats are biological).

Article Snippet: The following antibodies were used in this study: mouse anti-α-tubulin (T9026, 1:5000 for western blotting) from Sigma-Aldrich; rabbit anti-Septin6 (HPA005665, 1:4000 for western blotting) from Sigma-Aldrich, rabbit anti-SH3KBP1(Abclonal-A1952), mouse anti-GFP (M048-3, 1:4000 for western blotting and 1 uL antibody per milligram protein for Co-IP) from MBL International; anti-mouse (sc-516102, 1:4000) and anti-rabbit (sc-2004, 1:4000) horseradish peroxidase (HRP)-conjugated secondary antibodies from Santa Cruz Biotechnology.

Techniques: Concentration Assay, Expressing, Mutagenesis, Membrane, SDS Page, Staining, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Blebs regulate phosphoinositides distribution and promote cell survival through the Septin-SH3KBP1-PI3K axis

doi: 10.1101/2024.07.09.602823

Figure Lengend Snippet: (A)Protein–protein interaction (PPI) networks of PIK3R1 and Septin6 acquired through the STRING database( https://cn.string-db.org/ ). Thickness of the line indicate the degree of confidence prediction of the interaction.SH3KBP1 emerged as a potential linker between Septin6 and PIK3R1. (B)Representative images of GFP-Septin6 (upper left), PIK3R1-iRFP (upper right), SH3KBP1-mCherry-Cry2 (bottom left) and merged channel (bottom right) in MDA-MB-231 cell during cell blebbing induced by low concentration Latrunculin B. White arrows indicate Septin6-SH3KBP1-PIK3R1 colocalization at bleb neck. Scale bar, 5 μm. (C)Representative images of MDA-MB-231 cells expressing Opto-SH3KBP1 upon blue light exposure. Scale bar, 5 μm. Dashed boxes are zoomed in at the bottom right corner. Scale bar, 5 μm. (D)Left: representative images of MDA-MB-231 cells expressing Opto-FUS-IDR (upper) and PIK3R1-iRFP (bottom) upon blue light exposure. Right: representative images of MDA-MB-231 cells expressing Opto-SH3KBP1 (upper) and PIK3R1-iRFP (bottom) upon blue light exposure. White arrows indicate recruited PIK3R1 droplets by SH3KBP1. (E)Schematic representation of SH3KBP1 domain structure and ΔSH3-AB, SH3-AB, IDR(FUS)-ΔSH3-AB truncation design. (F)Representative images of Opto-ΔSH3-AB (upper) and Opto-SH3-AB (bottom) upon blue light exposure in PIK3R1-iRFP expressed MDA-MB-231 cells. Scale bar, 5 μm. (G)Left: co-immunoprecipitation assay showing interaction between PIK3R1-GFP and WT-SH3KBP1-Flag (upper) or SH3-AB-Flag (middle) or ΔSH3-AB-Flag (bottom) Right: co-immunoprecipitation assay showing interaction between Septin6 and WT-SH3KBP1-Flag (upper) or SH3-AB-Flag (middle) or ΔSH3-AB-Flag (bottom) (N = 3 experiments, repeats are biological). (H)Representative images of MDA-MB-231 cells expressing Opto-IDR(FUS)-ΔSH3-AB (upper) and PIK3R1-iRFP (bottom) upon blue light exposure. Scale bar, 5 μm. Dashed boxes are zoomed in (right). Scale bar, 5 μm.

Article Snippet: The following antibodies were used in this study: mouse anti-α-tubulin (T9026, 1:5000 for western blotting) from Sigma-Aldrich; rabbit anti-Septin6 (HPA005665, 1:4000 for western blotting) from Sigma-Aldrich, rabbit anti-SH3KBP1(Abclonal-A1952), mouse anti-GFP (M048-3, 1:4000 for western blotting and 1 uL antibody per milligram protein for Co-IP) from MBL International; anti-mouse (sc-516102, 1:4000) and anti-rabbit (sc-2004, 1:4000) horseradish peroxidase (HRP)-conjugated secondary antibodies from Santa Cruz Biotechnology.

Techniques: Concentration Assay, Expressing, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Blebs regulate phosphoinositides distribution and promote cell survival through the Septin-SH3KBP1-PI3K axis

doi: 10.1101/2024.07.09.602823

Figure Lengend Snippet: (A)Co-immunoprecipitation assay showing interaction between PIK3R1-Flag and Septin6 in WT and SH3KBP1-KD MDA-MB-231 cells. (N = 3 experiments, repeats are biological). (B)Representative surface renderings of PI(4,5)P2 and PI(3,4,5)P3 intensity in SH3KBP1-KD cells. (C)Time lapse images of MDA-MB-231 cells expressing PI(4,5)P2 sensor PLCδ-PH-mMaple3 (left) or PI(3,4,5)P3 sensor Akt-PH-mMaple3 (middle) after transient photoconversion at specific membrane sites in SH3KBP1-KD cells. Green dashed boxes are photoconverted sites. Other colored dashed boxes marked sensor intensity are calculated in right panel. Scale bar, 5 μm. (D)Co-immunoprecipitation assay showing interaction between Septin6 and PIK3R1-GFP in SH3KBP1-KD cells after FL-SH3BKP1-Flag rescue (upper) or ΔSH3-AB-Flag (middle) or SH3-AB-Flag (bottom) (N = 3 experiments, repeats are biological). (E)Representative surface renderings of PI(4,5)P2 (bottom) and PI(3,4,5)P3 (upper) intensity in SH3KBP1-KD cells after FL-SH3BKP1-Flag rescue (left) or ΔSH3-AB-Flag (middle) or SH3-AB-Flag (right). (F)A proposed model for bleb regulated PIs behavior: Septin-SH3KBP1-PI3K condensates localize at bleb neck and regulate PI(4,5)P2 transition to PI(3,4,5)P3, resulting in distinct PIs distribution on plasma membrane.

Article Snippet: The following antibodies were used in this study: mouse anti-α-tubulin (T9026, 1:5000 for western blotting) from Sigma-Aldrich; rabbit anti-Septin6 (HPA005665, 1:4000 for western blotting) from Sigma-Aldrich, rabbit anti-SH3KBP1(Abclonal-A1952), mouse anti-GFP (M048-3, 1:4000 for western blotting and 1 uL antibody per milligram protein for Co-IP) from MBL International; anti-mouse (sc-516102, 1:4000) and anti-rabbit (sc-2004, 1:4000) horseradish peroxidase (HRP)-conjugated secondary antibodies from Santa Cruz Biotechnology.

Techniques: Co-Immunoprecipitation Assay, Expressing, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

doi: 10.1073/pnas.2321338121

Figure Lengend Snippet: Fig. 1. Generation of deletion mutants in the promoter region of tim by CRISPR-cas9. (A) ChIP signal of CLK binding at tim in wild-type flies. CLK binds in two distinct E-box containing regions of the tim promoter: upstream of the transcriptional start site and within the second intron. CLK binding changes throughout the day peaking at ZT14. The direction of transcription is Right to Left. The y-axis scale is identical for all time points. (B) Description of tim promoter mutants generated by CRISPR-cas9. The deletion mutants are aligned to the wild-type (yw) genomic sequence. E-boxes are labeled in red, PER-boxes are labeled in blue, and TER-boxes are labeled in green. The deletions were named according to their position and size: tim_in24 (e.g., 24 bp deletion in the intron), tim_up10 (e.g., 10 bp deletion in the upstream region), tim_up122 and tim_up126. (C) RNA-sequencing results from fly heads show that there is a steady decrease in tim expression as the promoter deletions progress from mild to more extensive. Flies were collected at six timepoints throughout the day. Error bars represent the SD of two biological replicates. For tim_in24, tim_up10, we did one replication because of they only have very marginal effect on tim expression. The y-axis shows RPKM values (Reads Per Kilobase of transcript per Million mapped reads) normalized to the maximal level of mRNA during the day. (D) A representative western blot showing that TIM is significantly reduced in the tim_up126 compared to the wild-type control (yw). Flies were collected at six time points throughout the day. Beta-actin was used as a loading control. (E) Quantification of TIM levels in yw and tim_up126 as shown in D. TIM levels were normalized to beta-actin. Error bars represent the SD of two biological repeats.

Article Snippet: Blots were blocked in 5% milk in PBST (3.2 mM Na2 HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, and 0.05% Tween- 20, pH 7.4) for 1 h and incubated overnight with either rat anti- TIM at 1:4,000 dilution, rabbit anti- PER at 1:4,000 dilution or mouse anti- Actin (loading control; Santa Cruz) antibodies.

Techniques: CRISPR, Binding Assay, Generated, Sequencing, Labeling, RNA Sequencing, Expressing, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

doi: 10.1073/pnas.2321338121

Figure Lengend Snippet: Fig. 2. TIM represses clock gene transcription by affecting CLK DNA binding. (A–C) RNA sequencing was performed on both mutant and wild-type (yw) heads collected at six time points throughout the day. mRNA levels of per (A), vri (B) and pdp1 (C) at each time point were quantified and graphed to show the changes that occur throughout the day. For tim_up122, tim_up126, error bars represent the SD of two biological replicates. The y-axis shows RPKM values normalized to the maximal level of mRNA during the day. (D) ChIP results show CLK binding at the per locus in wild-type (yw) (Left; blue) and tim_up126 (Right; pink) across six time points throughout the day. In tim_up126, CLK binding to the per locus is shifted to earlier in the day.

Article Snippet: Blots were blocked in 5% milk in PBST (3.2 mM Na2 HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, and 0.05% Tween- 20, pH 7.4) for 1 h and incubated overnight with either rat anti- TIM at 1:4,000 dilution, rabbit anti- PER at 1:4,000 dilution or mouse anti- Actin (loading control; Santa Cruz) antibodies.

Techniques: Binding Assay, RNA Sequencing, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

doi: 10.1073/pnas.2321338121

Figure Lengend Snippet: Fig. 3. Immunostaining for TIM, PER, and PDP1 reveals that the components of the molecular clock are still properly expressed in the pacemaker neurons of the tim_ up126 mutant. (A–C) Quantification of the immunostaining signals of TIM (A), PER (B), and PDP1 (C) in yw (blue line) and tim_up126 (gray line). Flies were entrained in LD condition for 4 d before being subjected to constant darkness condition for 3 d. Brains were dissected for immunostaining at time points throughout the third day in constant darkness. Error bars represent the SEM.

Article Snippet: Blots were blocked in 5% milk in PBST (3.2 mM Na2 HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, and 0.05% Tween- 20, pH 7.4) for 1 h and incubated overnight with either rat anti- TIM at 1:4,000 dilution, rabbit anti- PER at 1:4,000 dilution or mouse anti- Actin (loading control; Santa Cruz) antibodies.

Techniques: Immunostaining, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

doi: 10.1073/pnas.2321338121

Figure Lengend Snippet: Fig. 4. The pacemaker neurons in tim_in24 and tim_up126 exhibit modestly reduced tim, while tim_up126 glia exhibit severely reduced tim. (A–C) Fluorescence in situ hybridization with RNAScope was performed to detect expression of tim mRNA in yw (A), tim_in24 (B), and tim_up126 (C) brains at ZT16. One representative max-Z projected brain hemisphere is shown per condition, along with representative Z-projected slices with no overlapping cells, demonstrating the punctate signal in the dorsolateral neurons (LNds) and ventrolateral neurons (LNvs) used for quantification in D. (D) Puncta quantification in Clock neurons (Top) and glia (Bottom) across conditions (four brains per genotype, two replicates with two brains each). Glia are defined by Repo signal, while Clock neurons are defined as cells outlined by tim signal with no repo signal. Statistical analysis was conducted with a one-way ANOVA with the post hoc Tukey HSD test.

Article Snippet: Blots were blocked in 5% milk in PBST (3.2 mM Na2 HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, and 0.05% Tween- 20, pH 7.4) for 1 h and incubated overnight with either rat anti- TIM at 1:4,000 dilution, rabbit anti- PER at 1:4,000 dilution or mouse anti- Actin (loading control; Santa Cruz) antibodies.

Techniques: Fluorescence, In Situ Hybridization, RNAscope, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

doi: 10.1073/pnas.2321338121

Figure Lengend Snippet: Fig. 6. CLK facilitates the accessibility of core clock gene regulatory regions. (A–D) UAS-CLK flies were crossed with Repo-GAL4; AGES-Gal80 and nSyb-Gal4; AGES- Gal80 to achieve adult-specific CLK expression. Two-week-old progeny were fed 10 mM auxin for 5 d while being entrained in LD condition. Flies were collected at ZT02 and ZT14 and 5,000 cells per replicate were used for tagmentation. ATAC-Seq tracks are aligned with tracks for CLK-ChIP (around the clock). (A and B) Adult-specific overexpression of CLK in glia results in a modest yet noticeable increment in chromatin accessibility within E-box regions of tim (A) and vri (B). (C and D) Adult-specific expression of CLK in neurons results in dramatically enhanced chromatin accessibility of the vri (C) and tim (D) genes. Both neural and glial-specific overexpression of CLK have at least two replicates. Purple arrows indicate regions of increased chromatin accessibility driven by CLK overexpression.

Article Snippet: Blots were blocked in 5% milk in PBST (3.2 mM Na2 HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, and 0.05% Tween- 20, pH 7.4) for 1 h and incubated overnight with either rat anti- TIM at 1:4,000 dilution, rabbit anti- PER at 1:4,000 dilution or mouse anti- Actin (loading control; Santa Cruz) antibodies.

Techniques: Expressing, Over Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

doi: 10.1073/pnas.2321338121

Figure Lengend Snippet: Fig. 5. Distinct chromatin accessibilities in the intronic and upstream E-Box regions of tim genome. (A) The results of ATAC-Seq analysis from glia (green) and clock neurons (blue) are shown for glial and neural marker genes—repo and nSyb. (B) The chromatin accessibilities of the tim regulatory region in glia, clock neurons and heads. Each track is consistent with three other replicates performed for the same cell type. Flies were entrained in LD condition at least for 3 d before the experiments. Approximately 5,000 cells for glia and clock neurons were collected by Fluorescence-activated cell sorting (FACS). For ATAC-Seq from heads was done using 10 heads per replicate. (C) The tim intronic E-box region has accessible chromatin in neural cells but not in glia since an early developmental stage (10 to 12 h after egg laying). Figure C was directly taken from the single-cell ATAC-Seq data published on http://shiny.furlonglab.embl.de/scATACseqBrowser/. Green and purple arrows for all figures indicate the tim126 peak and intronic peak respectively.

Article Snippet: Blots were blocked in 5% milk in PBST (3.2 mM Na2 HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, and 0.05% Tween- 20, pH 7.4) for 1 h and incubated overnight with either rat anti- TIM at 1:4,000 dilution, rabbit anti- PER at 1:4,000 dilution or mouse anti- Actin (loading control; Santa Cruz) antibodies.

Techniques: Marker, Fluorescence, FACS