Journal: Current biology : CB

Article Title: Growth at Cold Temperature Increases the Number of Motor Neurons to Optimize Locomotor Function

doi: 10.1016/j.cub.2019.04.072

Figure Lengend Snippet: (A) RT-PCR for trpm8 (236 bp) from cDNA extracted from stage 24 whole embryo or dissected spinal cord. +/− RT, in the presence or absence of the reverse transcriptase, respectively, during conversion of isolated mRNA into cDNA. (B) In situ hybridization for trpm8 in stage 24 embryos showing specific labeling in brain and spinal cord. (C) Western blot assays from egg, stage 24 wild-type whole embryo or from stage 40 whole larva, control morpholino (MO) or TRPM8-translation-blockingMO 1 (TRPM8-tbMO1) lysates. Predicted TRPM8 molecular weight [MW]: 132 kDa. Shown are representative examples of one of 3 independent experiments. β-tubulin was used as loading control. (D) Immunostained transverse section of stage 25 spinal cord (outlined). D, dorsal; V, ventral; scale bar, 20 μm; arrows indicate TRPM8 clusters in ventral neuron domains. NCAM labeling was used as counterstaining. (E) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged at 1 Hz for 90 s. Either 100 μM (−)-menthol or vehicle (0.05% DMSO)was added after 35 s of imaging and recording continued for another 60 s. Images show a menthol-responsive ventral neuron before (left, control) and after (right) addition of (−)-menthol. Colored scale shows fluorescence intensity in arbitrary units. Traces show the changes in fluorescence for the indicated cell (arrow) in both trials. (F) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged in 30-min intervals at cold (14.5°C) and warm (26.5°C) temperatures in the absence (vehicle, 0.1% DMSO) or presence of 10 μM AMTB, TRPM8 inhibitor. Scatterplots show changes in Ca 2+ spike frequency when switching temperatures in individual spinal neurons and geometric mean (black lines) from N = 3 ventral spinal cords per condition (n of neurons analyzed: DMSO, 62; AMTB, 71). Teal circles represent neurons with higher spike frequency at 14.5°C, magenta circles represent neurons with higher spike frequency at 26.5°C, and black circles represent neurons with no change in spike frequency across temperatures; ****p < 0.0001, comparison within treatments Wilcoxon matched-pairs signed rank, two-tailed test. (G) RT-PCR from cDNA collected from stage 46 larvae previously injected with 2.5 pmol standard control morpholino (Control-MO) or TRPM8-splicing-blocking morpholino (TRPM8-sbMO) shows that trpm8 mature transcript (349 bp) is not detected in TRPM8-sbMO animals. odc : ornithine decarboxylase (101 bp) as positive control. (H) TRPM8-sbMO or Control-MO containing spinal cord from stage 24 embryos were Ca 2+ -imaged for 30 min at cold temperature (14.5°C). Graph shows individual (scatterplots) and geometric mean (black lines) Ca 2+ spike frequency from N = 3 ventral spinal cords pergroup (n of neurons analyzed: Control-MO, 81; TRPM8-sbMO, 64), ****p < 0.001, Kolmogorov-Smirnov, two-tailed test. See also and .

Article Snippet: For protein loading control, PVDF membranes were stripped in stripping buffer (0.2 M glycine-HCl buffer, pH 2.5, 0.05% Tween) for 20 min then re-probed overnight with anti-β-tubulin mouse monoclonal antibody (1:50 in 5% milk, Developmental Studies Hybridoma Bank), followed by HRP-conjugated secondary antibody (Jackson ImmunoResearch; 1:10,000) and visualized as described above.

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Isolation, In Situ Hybridization, Labeling, Western Blot, Control, Molecular Weight, Imaging, Fluorescence, Comparison, Two Tailed Test, Injection, Blocking Assay, Positive Control

Journal: Current biology : CB

Article Title: Growth at Cold Temperature Increases the Number of Motor Neurons to Optimize Locomotor Function

doi: 10.1016/j.cub.2019.04.072

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For protein loading control, PVDF membranes were stripped in stripping buffer (0.2 M glycine-HCl buffer, pH 2.5, 0.05% Tween) for 20 min then re-probed overnight with anti-β-tubulin mouse monoclonal antibody (1:50 in 5% milk, Developmental Studies Hybridoma Bank), followed by HRP-conjugated secondary antibody (Jackson ImmunoResearch; 1:10,000) and visualized as described above.

Techniques: Recombinant, Western Blot, In Situ, Luciferase, Reporter Assay, Reverse Transcription, cDNA Synthesis, Isolation, Sequencing, Control, Knockdown, Software

Journal: STAR Protocols

Article Title: Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture

doi: 10.1016/j.xpro.2022.101549

Figure Lengend Snippet:

Article Snippet: Incubate neurons with primary antibodies (PI3P, Z-P003, Echelon, 1:50, PI4P, Z-P004, Echelon, 1:200, or PI(4,5)P 2 , Z-A045, Echelon, 1:150 and Rab8, R5530, Sigma-Aldrich, 1:150 or TGN46, ab50595, Abcam, 1:200) in buffer A containing 5% NGS for 1 h. 41.

Techniques: Recombinant, Electron Microscopy, Software, Microscopy, Cell Culture, In Vitro, Cell Counting

Journal: eLife

Article Title: Thalamocortical axons control the cytoarchitecture of neocortical layers by area-specific supply of VGF

doi: 10.7554/eLife.67549

Figure Lengend Snippet:

Article Snippet: After blocking with PBS containing 5% normal goat/donkey serum and 0.1% Triton X-100 (blocking buffer) at RT for 1 hr, the sections were incubated at 4°C overnight with the following antibodies in the blocking buffer: mouse anti-RORC (1:800; catalog no. PP-H3925-00, Perseus Proteomics; although this antibody recognizes RORα, β and γ, RORγ is not expressed in the postnatal brain, and RORα expression generally overlaps with RORβ but is weak in the cortex [in Allen Brain Atlas]), rabbit anti-GFP (1:800; #A6455, Invitrogen), rabbit anti-Iba1 (1:500; catalog no. 019-19741, Wako), rabbit anti-5HTT (1:10,000; catalog no. 24330, ImmunoStar), goat anti-Brn2 (1:50; catalog no. sc-6029, Santa Cruz), rat anti-Ctip2 (1:200; catalog no. ab18465, Abcam), rabbit anti-Tbr1 (1:500; catalog no. ab31940, Abcam), rabbit anti-RFP (1:1000; catalog no. PM005, MBL), mouse anti-FLAG (1:1000, catalog no. F1804, Sigma-Aldrich), rabbit anti-ssDNA (1:300; catalog no. 18731, MBL), rabbit anti-RORβ (1:5000; catalog no. pAb-RORβHS-100, Diagenode), mouse anti-NeuN (1:400; catalog no. MAB377, Chemicon), rabbit anti-Cux1 (1:100, catalog no. sc-13024, Santa Cruz), rabbit anti-NRN1 (1:50; catalog no. sc-25261, Santa Cruz), and goat anti-VGF (1:50; catalog no. sc-10381, Santa Cruz).

Techniques: CRISPR, Recombinant, Plasmid Preparation, Expressing, Sequencing, Imaging, Software, Staining

Journal: iScience

Article Title: Mapping transcriptional heterogeneity and metabolic networks in fatty livers at single-cell resolution

doi: 10.1016/j.isci.2022.105802

Figure Lengend Snippet:

Article Snippet: ImmPRESS® HRP Horse Anti-Mouse IgG Polymer Detection Kit , Vector laboratories , Cat# MP-7402.

Techniques: Virus, Recombinant, Saline, Staining, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Activity Assay, Polymer, Sequencing, Expressing, Software, Imaging, Cell Culture, Microscopy, Blocking Assay

Journal: Cancer cell

Article Title: Mitochondrial reprogramming underlies resistance to BCL-2 inhibition in lymphoid Malignancies

doi: 10.1016/j.ccell.2019.08.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Immunohistochemistry and image acquisition Immunohistochemical staining (IHC) for MCL1 (Clone 22, 1:100, Santa Cruz), Acetyl-CoA Carboxylase (Clone C83B10, 1:50, Cell Signaling), Phospho-Acetyl-CoA Carboxylase (Clone D7D11, 1:50, Cell Signaling) or AMPKα (clone D5A2, 1:50, Cell Signaling) was performed using an automated staining system (BondRX, Leica Biosystems, Buffalo Grove, IL) according to the manufacturer’s protocol and as previously described ( Roemer et al., 2016 ).

Techniques: Recombinant, Cell Viability Assay, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Picogreen Assay, RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction, CRISPR, Knock-Out, Software

Journal: Cell metabolism

Article Title: Telomere dysfunction induces sirtuin repression that drives telomere-dependent disease

doi: 10.1016/j.cmet.2019.03.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After washing in PBS, slides were incubated with ImmPRESS HRP Anti-Mouse IgG Polymer (Vector Laboratories) at room temperature for 1 hour.

Techniques: Construct, Plasmid Preparation, Recombinant, Protease Inhibitor, Bradford Assay, Fractionation, Reporter Assay, Mutagenesis, Activity Assay, Staining, In Situ, Knock-Out, Transgenic Assay, Software, RNA Sequencing Assay

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) Joint swelling and clinical scores in wild-type (WT), Fas lpr/lpr , Fasl gld/gld , and Fas –/– mice (n = 6 per group). ( B ) Joint swelling and clinical scores in WT, Fasl Δm/Δm , Fasl Δs/Δs , and Fasl Δs/Δs mice injected with sFasL (n = 6 per group). ( C , D ) Gross and microscopic examination of arthritis (magnified 10× in the upper panel and 200× in the lower panel). Scale bars: 1 cm ( C ), 200 μm ( D , upper panel), and 100 μm ( D , lower panel). ( E ) Tandem mass spectra of unique DR5 peptides. ( F ) Transcript levels of Tnfrsf10b in synovial CD45 + immune cells and CD45 – non-immune cells from WT mice with or without AIA. ( G ) Immunohistochemistry of DR5 expression in joint tissue from a healthy control subject and a patient with rheumatoid arthritis (n = 3; magnified 400×, scale bar: 50 μm). ( H ) Flow cytometric analysis of biotinylated protein binding to EL4 cells transfected with human WT TNFRSF10B preincubated with recombinant hTRAIL, or simultaneously incubated with anti-FasL, or anti-DR5 antibodies. ( I ) Flow cytometric analysis of biotinylated FasL binding on hFLSCs with FAS and/or TNFRSF10B knockout, and TNFRSF10B and/or FAS overexpression in FAS and TNFRSF10B double knockout (DKO) cells. ( J ) hLFSCs were preincubated with TNF-α (as a negative control), FasL, or TRAIL and cross–linked with BS 3 . Lysates from these cells were immunoprecipitated with anti–DR5 or control IgG antibody and immunoblotted with anti-DR5, TNF-α, FasL, or TRAIL antibodies. ( K ) Flow cytometric analysis of DR5–Fc binding on EL4 cells transfected with human WT FASLG in the presence of recombinant hTRAIL, anti-DR5, or FasL antibodies. Data were pooled from three ( A , B , and D–G ) or four ( H, K ) independent experiments and are presented as mean ± standard error of the mean (SEM). *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way analysis of variance (ANOVA). Figure 1—source data 1. Numerical data obtained during experiments represented in , , and .

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Injection, Immunohistochemistry, Expressing, Control, Protein Binding, Transfection, Recombinant, Incubation, Binding Assay, Knock-Out, Over Expression, Double Knockout, Negative Control, Immunoprecipitation

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) Schematic diagram showing AP–MS analyses. ( B ) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of biotinylated Fc (control) or sFasL–Fc cross-linked protein complexes. The fractions used in in-gel digestion are separated by red lines. ( C ) Expression of DR5 in CD45 + and CD45 – cells from the joint tissues of WT mice with arthritis. ( D ) Flow cytometry analyses of the expression of Fas and DR5 in human (h) fibroblast-like synovial cells (FLSCs). ( E ) Flow cytometry analyses of FasL–Fc binding to hFLSCs in the presence of anti-DR5 and/or anti-Fas antibodies. ( F , G ) Flow cytometry analyses of human DR5 ( F ) and Fas ( G ) expression in EL4 cells transfected with human WT tumor necrosis factor receptor superfamily (TNFRSF)10B ( F ) and FAS ( G ). ( H ) Expression of TNFRSF10B and FAS in EL4 mouse T cells transfected with various human genes. ( I ) Flow cytometry analyses of biotinylated protein binding to EL4 cells transfected with human WT FAS preincubated with recombinant human (h) TNF-related apoptosis-inducing ligand (TRAIL) or treated simultaneously with anti-Fas and DR5 antibodies. ( J ) Flow cytometry analyses of FasL–Fc binding to hFLSCs after preincubation with recombinant sTRAIL or sFasL. All experiments were performed four times independently.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Protein-Protein interactions, Polyacrylamide Gel Electrophoresis, Control, Expressing, Flow Cytometry, Binding Assay, Transfection, Protein Binding, Recombinant

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) hFLSCs and mouse synovial fibroblasts were stimulated with human or mouse sFasL in the presence or absence of anti-mouse Fas or anti-DR5 antibodies, as well as anti-human Fas or anti-DR5 antibodies for 24 hr. CX3CL1 levels in culture supernatants were measured using ELISA. ( B , C ) Jurkat cells ( B ) or mouse splenocytes ( C ) were incubated for 24 hr with recombinant FasL and TRAIL in the presence or absence of human or mouse anti-DR5 or anti-Fas antibodies. Jurkat and gated splenic TCRβ + CD4 + T cell death was measured using flow cytometry. ( D ) Jurkat cell death was measured using flow cytometry after FasL or FasL–Fc treatment in the presence or absence of anti-Fas or anti-DR5 antibodies for 24 hr. Data are presented as mean ± SEM. All experiments were performed three times independently. *p<0.05; **p<0.01. Data were analyzed using one-way ANOVA.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Flow Cytometry

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) Flow cytometry analyses of FasL–Fc binding to hFLSCs that were knocked down with FAS , TNFRSF10B , TNFRSF1A , TNFRSF10A , or TNFRSF12 . ( B ) Expression of FAS , TNFRSF10B , TNFRSF10A , TNFRSF1A , and TNFRSF12 in siRNA-transfected hFLSCs. ( C ) Expression of FAS and TNFRSF10B in FAS - and/or TNFRSF10B -knockout (KO) cells and KO cells transfected with TNFRSF10B and/or FAS overexpression vector. ( D , E ) Expression of Fas and DR5 ( D ) and biotinylated TRAIL binding ( E ) in FAS - and/or TNFRSF10B -KO hFLSCs and DKO (DR5 and Fas gene double knockout) cells transfected with TNFRSF10B and/or FAS in expression vectors. Biotinylated TRAIL binding was quantified by streptavidin (sAv)–fluorescein isothiocyanate staining intensity using flow cytometry. ( F, G ) Surface plasmon resonance assays for DR5–FasL ( F ) and DR5–TRAIL ( G ) interactions. ( H ) hFLSCs were incubated with PBS (control), 6× His-tagged FasL, or 6× His-tagged TRAIL and cross-linked using BS 3 . Cell lysates were immunoprecipitated with anti-His or control IgG antibodies and then immunoblotted with anti-Fas, DR5, TRAIL, and FasL antibodies. ( I ) FASLG , TNFSF10 , and TNF expression in EL4 mouse T cells transfected with human genes. ( J ) Flow cytometry analyses of DR5–Fc and Fas–Fc binding to EL4 cells transfected with human WT FASLG in the presence of anti-FasL antibodies. ( K, L ) Flow cytometry analyses of human FasL, TNF-α, and TRAIL expression ( K ), as well as DR5–Fc and Fas–Fc binding to EL4 cells transfected with human WT FASLG , TNFA , or TRAIL ( L ) and gated on transfected cells expressing the target proteins. Data were pooled from three independent experiments and are presented as mean ± SEM (n = 4 in B ). **p<0.01; ****p<0.0005. Data were analyzed using one-way ANOVA.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Flow Cytometry, Binding Assay, Expressing, Transfection, Knock-Out, Over Expression, Plasmid Preparation, Double Knockout, Staining, SPR Assay, Incubation, Control, Immunoprecipitation

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A , B ) FasL–Fc binding to hFLSCs or EL4 cells transfected with human FAS , or TNFRSF10B after preincubation with human sTRAIL or sFasL. ( C ) Model of FasL and DR5 derived from the crystal structure of the FasL/DcR3 complex (Protein Data Bank: 4 MSV) and TRAIL/DR5 complex (Protein Data Bank: 1D4V). ( D , E ) Flow cytometric analysis of FasL–Fc or DR5–Fc binding to EL4 cells transfected with human WT or mutated TNFRSF10B or FASLG . ( F , G ) Comparison of the effects of sFasL and sTRAIL on ( F ) apoptosis and ( G ) necroptosis in hFLSCs. ( H ) Joint swelling and clinical scores in Fasl gld/gld mice injected with Z–VAD–FMK and/or sFasL (n = 6 per group). Data were pooled from four ( A , B , and D–G ) or three ( H ) independent experiments and are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA. Figure 2—source data 1. Numerical data obtained during experiments represented in , .

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Binding Assay, Transfection, Derivative Assay, Comparison, Injection

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) hLFSCs were preincubated with FasL and TRAIL using an excess of TRAIL in lane 3 (TRAIL [4 μg/mL]+FasL) and an excess of FasL in lane 6 (FasL [4 μg/mL]+TRAIL) before cross-linking with BS 3 . Lysates from these cells were immunoprecipitated with anti-DR5 (lanes 2, 3, 5, and 6) or control IgG (lanes 1 and 4) antibodies and immunoblotted with anti-DR5, FasL, or TRAIL antibodies. ( B ) Crystal structures of the TRAIL/DR5 (Protein Data Bank: 1D4V) and FasL/DcR3 (Protein Data Bank: 4 MSV) complexes. ( C ) Alignment of the human DcR3 and DR5 as well as the human FasL and TRAIL sequences. The point mutations in the mutant huDR5–cysteine-rich domains (CRD)two and CRD3, the mutant FasL–CRD2 interacting domain, and the mutant FasL–CRD3 interacting domain are indicated by asterisks (*). ( D ) Flow cytometry analyses of human DR5 in EL4 cells transfected with human WT or mutant TNFRSF10B . ( E ) Flow cytometry analyses of human FasL in EL4 cells transfected with human WT or mutant FASLG . Experiments ( A , D , and E ) were performed three times independently.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Immunoprecipitation, Control, Mutagenesis, Flow Cytometry, Transfection

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A, B ) Microarray assay using joint tissues from WT, Fas lpr/lpr , and Fasl gld/gld mice with arthritis. ( C ) Cx3cl1 transcript levels estimated in joint tissues from WT, Fas lpr/lpr , Fas –/– , Fasl gld/gld , Fasl Δs/Δs , and Tnfrsf10b KO mice with arthritis. ( D ) Cx3cl1 expression in CD45 + immune and CD45 – non–immune cells from the joints of WT mice with arthritis after sFasL treatment. ( E, F ) CX3CL1 transcript levels estimated in hFLSCs in the presence of anti-Fas and/or anti-DR5 antibodies ( E ) and FAS (Fas), TNFSF10B (DR5), or FAS , and TNFRSF10B DKO, or TNFRSF10B and FAS overexpression in DKO hFLSCs ( F ). ( G ) Cx3cl1 expression in synovial fibroblasts from WT, Fas lpr/lpr , Fas –/– , or Tnfrsf10b KO mice in the presence or absence of sFasL. ( H, I ) CX3CL1 transcript levels estimated after sFasL stimulation in hFLSCs in the presence of MEK (U0126), ERK (PD980259), p38 kinase (SB203580), and NF-κB (MG132 and BMS345541) inhibitors ( H ) or transfection with control, RELA , CHUK (IKKa), or IKBKB (IKKb) siRNA ( I ). ( J ) Synovial fibroblasts obtained from WT mice with arthritis were incubated with sFasL or sTRAIL and CX3CL1 levels were measured using ELISA. ( K ) hFLSCs were stimulated with sFasL after preincubation with various concentrations of sTRAIL for 30 min and CX3CL1 levels were measured in the culture supernatant. Data were pooled from three ( C–G and K ) or four ( H–J ) independent experiments and are presented as mean ± SEM (n = 4 for C–K ). *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA. Figure 3—source data 1. Numerical data obtained during experiments represented in , and .

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Microarray, Expressing, Over Expression, Transfection, Control, Incubation, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) CX3CL1 was measured in culture supernatants of CD45 + immune and CD45 – non-immune cells from the joints of WT mice with arthritis after sFasL stimulation. ( B–H ) Cx3cl1 transcripts and CX3CL1 protein in culture supernatants were estimated in human ( B–F ) and mouse ( G, H ) FLSCs after stimulation with sFasL or FasL–Fc in the presence of anti-Fas or anti-DR5 antibodies. ( I ) CX3CL1 transcript of hFLSCs transfected with control, TNFRSF1A , FAS , TNFRSF12 , TNFRSF10A , and TNFRSF10B siRNA was measured after stimulation with sFasL. ( J ) FAS and/or TNFRSF10B KO hFLSCs, and DKO hFLSCs were transfected with TNFRSF10B. FAS in the expression vector was stimulated with sFasL. CX3CL1 levels were measured in the culture supernatants. ( K ) Levels of CX3CL1 in culture supernatants of synovial fibroblasts obtained from WT, Fas lpr/lpr , Fas –/– , or Tnfrsf10b KO mice with arthritis after incubation with sFasL. ( L , M ) Levels of CX3CL1 in culture supernatants of hFLSCs after sFasL stimulation for 2 hr in the presence of MEK (U0126), ERK (PD980259), p38 kinase (SB203580), and NF-κB (MG132, and BMS345541) inhibitors ( L ) or transfection with control, RELA , CHUK (IKKa), or IKBKB (IKKb) siRNA ( M ). ( N ) Blotting assay for components of the NF-κB signaling pathway in hFLSCs stimulated with sFasL for the durations indicated, all preincubated with anti-Fas antibodies. ( O , P ) CX3CL1 transcript ( O ) and CX3CL1 protein ( P ) levels in culture supernatants from hFLSCs after stimulation with sFasL in the presence of 50 μM Z–VAD–FMK. Data were pooled from four ( A–F , L , and M ) or three ( G–K , O , and P ) independent experiments and are presented as mean ± SEM (n = 5; A–M , O , and P ). NS, not significant; *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Incubation

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) Joint swelling and clinical scores in WT mice injected with anti-DR5 or anti-Fas antibodies to measure AIA (n = 5 per group). ( B, C ) Joint swelling and clinical scores in WT and Tnfrsf10b KO mice injected with sFasL or phosphate-buffered saline (PBS) to measure AIA (n = 5 per group). ( D ) Cx3cl1 transcript levels in the joints were estimated in WT, Tnfrsf10b KO, Fasl Δs/Δs , and Fasl Δm/Δm mice injected with sFasL or PBS to measure AIA (n = 5). ( E, F ) Joint swelling and clinical scores ( E ), and transcript levels of various cytokines and chemokines in joint tissues of Tnfrsf10b KO mice injected with CX3CL1 or PBS to measure AIA ( F ) (n = 6 per group). ( G ) Joint swelling and clinical scores of WT and Cx3cr1 KO mice in the presence or absence of sFasL to measure AIA (n = 6 per group). Data were pooled from three independent experiments and are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA. Figure 4—source data 1. Numerical data obtained during experiments represented in , .

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Injection, Saline

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A, B ) Joint swelling and clinical scores together with transcript levels of various cytokines and chemokines in joint tissues from Fasl gld/gld mice injected with sFasL, as well as anti-Fas, or anti-DR5 antibodies (n = 6 per group). ( C, D ) Joint swelling and clinical scores in Fasl Δs/Δs ( C ) and Fasl gld/gld ( D ) mice injected with CX3CL1 or PBS. ( E, F ) Transcript levels of various cytokines and chemokines in joint tissues from Fasl Δs/Δs ( E ) or Fasl gld/gld ( F ) mice injected with CX3CL1 or PBS to measure AIA (n = 6 per group). Data were pooled from four ( A, B ) or three ( C–F ) independent experiments and are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Injection

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet:

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Immunohistochemistry, Cytometry, Purification, Recombinant, In Vitro, Western Blot, In Vivo, Injection, Neutralization, Control, Cell Isolation, Enzyme-linked Immunosorbent Assay, Gene Expression, Negative Control, Staining, Sequencing, Software

Journal: STAR Protocols

Article Title: XACT-seq: A photocrosslinking-based technique for detection of the RNA polymerase active-center position relative to DNA in Escherichia coli

doi: 10.1016/j.xpro.2021.100858

Figure Lengend Snippet:

Article Snippet: Add 50 μL of 1 × Taq DNA polymerase buffer (NEB) to wash the beads and to prepare samples for the next step.

Techniques: Recombinant, Staining, Purification, Sequencing, Plasmid Preparation, Software, Electroporation

Journal: STAR Protocols

Article Title: XACT-seq: A photocrosslinking-based technique for detection of the RNA polymerase active-center position relative to DNA in Escherichia coli

doi: 10.1016/j.xpro.2021.100858

Figure Lengend Snippet:

Article Snippet: Add 50 μL of 1 × Taq DNA polymerase buffer (NEB) to wash the beads and to prepare samples for the next step.

Techniques: Concentration Assay

Journal: The EMBO Journal

Article Title: Acidity suppresses CD8 + T-cell function by perturbing IL-2, mTORC1, and c-Myc signaling

doi: 10.1038/s44318-024-00235-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The 293T and C1498 cell lines (ATCC) were grown in DMEM medium containing 4.5 g/L glucose, sodium pyruvate, and glutamax (Gibco), supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 10 mM HEPES, 50 U/mL penicillin, and 50 μg/mL streptomycin (Gibco).

Techniques: CRISPR, Recombinant, Plasmid Preparation, Sequencing, Staining, Lysis, Software, Cell Isolation, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Real-time Polymerase Chain Reaction

Journal: Cell

Article Title: Multi-level Proteomics Identifies CT45 as a Chemosensitivity Mediator and Immunotherapy Target in Ovarian Cancer

doi: 10.1016/j.cell.2018.08.065

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A complete list of cell lines can be found in the . table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal Ki-CT45-2 antibody Dr. Hans-Jürgen Heidebrecht (Kiel, Germany) N/A Mouse monoclonal anti-HLA-ABC antibodies W6/32 Thermo Fisher Scientific #MA1-19027; RRID: AB_1076699 Rabbit monoclonal anti-γH2AX Cell Signaling #9718; RRID: AB_2118009 Rabbit polyclonal anti-Cleaved CASP-3 Cell Signaling #9661; RRID: AB_2341188 Rabbit anti-IgG-HRP Cell Signaling #7074; RRID: AB_2099233 Mouse anti-IgG-HRP Cell Signaling #7076; RRID: AB_330924 Rabbit polyclonal anti-PPP4C Bethyl #A300-835A; RRID: AB_597901 Rabbit polyclonal anti-SMEK2 Bethyl #A300-842A; RRID: AB_597905 Rabbit polyclonal anti-KAP1 Bethyl #A300-274A; RRID: AB_185559 Mouse monoclonal anti-Actin Sigma #A5441; RRID: AB_476744 Rabbit polyclonal anti-CT45A Sigma #SAB1301842 Rabbit polyclonal anti-PPP4R2 Sigma #HPA034695; RRID: AB_10671231 Rabbit polyclonal anti-PPP4R3α Sigma #HPA002568; RRID: AB_1079207 Rabbit monoclonal anti-FANCD2 Abcam #ab108928; RRID: AB_10862535 Mouse monoclonal anti-γH2AX Life Technologies #MA1-2022; RRID: AB_559491 Mouse monoclonal anti-V5 Life Technologies #MA5-15253; RRID: AB_10977225 Mouse monoclonal anti-GST Abcam #3G10/1B3; RRID: AB_524046 Biological Samples Ovarian cancer specimens, Tables S1 and S2 This paper N/A Tumor infiltrating T cells This paper N/A Chemicals, Peptides, and Recombinant Proteins Carboplatin Hospira N/A Growth factor reduced Matrigel BD Biosciences (Rockville, MD) 356231 Synthetic HLA-I peptides of CT45 Core facility, Max Planck Institute of Biochemistry, Martinsried, Germany N/A MHC class-I tetramers NIH Tetramer Core Facility N/A Recombinant PP4R3β Dr. Jakob Nilsson (University of Copenhagen, Denmark) N/A Recombinant GST-CDC20 Dr. Jakob Nilsson (University of Copenhagen, Denmark) N/A Critical Commercial Assays Ser/Thr phosphatase assay kit Millipore #17-127 Deposited Data Raw and analyzed data This paper http://proteomecentral.proteomexchange.org/cgi/GetDataset (dataset identifier PXD010372) Patient information Tables S1 and S2 This paper N/A Ovarian cancer proteome data, Table S3 This paper N/A Patient information of tissue microarray, Table S4 This paper N/A HLA-I peptidomes of cell lines, Table S5 This paper N/A RNaseq analysis of 37 tissues Uhlén et al., 2015 https://www.proteinatlas.org/about/download RNAseq expression analysis of HGSOC and across cancers TCGA IlluminaHiseq BC and HiSeqV2_PANCAN Experimental Models: Cell Lines SKOV3ip1, female origin.

Techniques: Recombinant, Phosphatase Assay, Microarray, Expressing, Plasmid Preparation, Sequencing, Software

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Acetyl-Histone H3 (ChIP: 1/50) , Cell Signaling Technology , 9649.

Techniques: Recombinant, Sequencing, Control, Transfection, Membrane, Reverse Transcription, Software, Microarray, Subcloning, shRNA, Cell Viability Assay, Viability Assay, Reporter Assay, In Situ, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Extraction, Bicinchoninic Acid Protein Assay, Magnetic Beads, Gel Extraction, Purification, Ligation, Mutagenesis, Chromatin Immunoprecipitation