Journal: Molecular Cell

Article Title: DNAJC9 integrates heat shock molecular chaperones into the histone chaperone network

doi: 10.1016/j.molcel.2021.03.041

Figure Lengend Snippet: J domain mutation traps DNAJC9 on chromatin genome-wide in a histone-dependent manner (A) DNAJC9 domain map with relevant mutations. (B) Western blots of soluble and chromatin extracts from cells expressing DNAJC9-MYC-FLAG WT, J, or 4AJ mutants compared with control cells. See also Figure S4 . (C–E) Quantitative ChIP-seq of cells expressing DNAJC9-MYC-FLAG WT, J, or 4AJ mutants compared with control cells. ChIP-seq reads were quantitated in 10 kb windows with a 5 kb step. Plots represent data averaged from n = 2 biological replicates. (C) Visualization of spike-in normalized ChIP-seq signal in DNAJC9 WT, J, 4AJ, and control samples, quantitated with reference-adjusted reads per million (RRPM), and raw input reads over the region depicted. (D) Boxplots of spike-in normalized DNAJC9 ChIP-seq signal across the genome quantitated with reference-adjusted reads per million (RRPM), Log 2 (n + 1). Black line, median; whiskers, 1.5 × interquartile range. (E) Boxplots of input corrected signal for DNAJC9 J mutant over gene bodies and intergenic regions (left) or gene bodies parsed to active and inactive genes (right). Black line, median; whiskers, 1.5 × interquartile range.

Article Snippet: Raw Data for Western Blots, Gel-based Assays and ITC , This study , Mendeley Data: https://doi.org/10.17632/y9mgyw9r59.1.

Techniques: Mutagenesis, Genome Wide, Western Blot, Expressing, Control, ChIP-sequencing

Journal: Molecular Cell

Article Title: DNAJC9 integrates heat shock molecular chaperones into the histone chaperone network

doi: 10.1016/j.molcel.2021.03.041

Figure Lengend Snippet: DNAJC9-directed HSP70 activity facilitates histone supply to and transactions within chromatin (A–D) DNAJC9 WT, mutant, and control purifications from soluble and chromatin extracts subjected to label-free mass spectrometry analysis (n = 6 biological replicates). Proteins are referred to by human UniProt protein identification code. See also and and . (A) Bubble plots from (left) soluble and (right) chromatin fraction purifications showing enrichment (red) and depletion (blue) in J domain and histone binding mutants (J and 4A, respectively) compared with WT. Ratios calculated from bait-normalized LFQ intensities (LFQ B.N. ). Data analysis steps are detailed in and . (B) Left: Euclidean clustering analysis of soluble fraction LFQ B.N. intensities for factors identified in at least six of six experiments for WT, J, or 4A mutants and additionally significantly enriched in at least one set over the control purifications (S0 = 2, FDR = 0.01). Region of interest highlighted (black box) and magnified (right) to show trends of enrichment and depletion of histones, ribosomal proteins, HSP70s and other factors. (C) Western blots of soluble extracts from cells expressing DNAJC9 WT, J, or 4A mutants and control cells showing the accumulation of soluble histones in cells expressing the DNAJC9 J mutant. (D) STRING-db network of factors most significantly enriched in DNAJC9 WT purifications and overlaid ratios of enrichment/depletion in J and 4A mutants. Sixteen of 22 nodes with Log 2 (LFQ WT/C) B.N. > 1.8 were connected to each other at a STRING confidence level of 0.6; red edges represent nodes connected with experimental evidence.

Article Snippet: Raw Data for Western Blots, Gel-based Assays and ITC , This study , Mendeley Data: https://doi.org/10.17632/y9mgyw9r59.1.

Techniques: Activity Assay, Mutagenesis, Control, Mass Spectrometry, Binding Assay, Western Blot, Expressing

Journal: Molecular Cell

Article Title: DNAJC9 integrates heat shock molecular chaperones into the histone chaperone network

doi: 10.1016/j.molcel.2021.03.041

Figure Lengend Snippet:

Article Snippet: Raw Data for Western Blots, Gel-based Assays and ITC , This study , Mendeley Data: https://doi.org/10.17632/y9mgyw9r59.1.

Techniques: Virus, Recombinant, Sequencing, Modification, Purification, Mass Spectrometry, Western Blot, Negative Control, Software, Magnetic Beads, Immunoprecipitation

Journal: Cell reports

Article Title: Steroid hormone catabolites activate the pyrin inflammasome through a non-canonical mechanism

doi: 10.1016/j.celrep.2022.111472

Figure Lengend Snippet: (A) Monocytes from HDs (n = 2–12) were treated with colchicine or nocodazole and 30 min later with the indicated stimuli. Propidium iodide incorporation was monitored every 5 min for 3 h. (B) The area under the cure (AUC) is shown. For each HD, the AUC values in the presence of inhibitors were normalized to the AUC value obtained in the absence of inhibitor. (C) Monocytes from HDs (n = 8–14) were treated with colchicine and 30 min later with the indicated stimuli. IL-1β concentration in the supernatant was quantified at 3 h post-addition. (D) RhoA activity was determined by G-LISA at different time post-treatment in the lysate of U937 cells. The activity of the different treatments at the indicated time is presented in . (E) Doxycycline-induced, PMA-differentiated U937 macrophages expressing MEFV Full-length (FL), deleted of the pyrin (ΔPYD), of the phosphorylated linker (ΔPLD), of the BBox (ΔBbox), of the coiled-coil (ΔCC), or the B30.2 (ΔB30.2) domains were treated with LPS for 3 h and then with the indicated stimuli. IL-1β concentration in the supernatant was quantified at 3 h post-addition. (F) Doxycycline-induced, U937 monocytes expressing WT or ΔB30.2 MEFV were treated with the indicated stimuli for 90 min. Pyrin S242 phosphorylation was assessed by western blot analysis following immunoprecipitation. “*” indicates a cleaved form of pyrin. (A–E) Mean and SEM are shown. (B and C) Each dot represents the value for one HD. (D and E) One experiment with technical triplicates representative of two (D) to three (E) independent experiments is shown. (B–C) Wilcoxon matched-pairs signed rank tests were performed to compare values with or without colchicine/nocodazole. Two-tailed p values: (B) **p = 0.0078, ***p < 0.001; (C) ***p < 0.001, **p = 0.002; (D) ordinary one-way ANOVA with Holm-Sidak’s correction for multiple tests was performed. *p = 0.015, ***p < 0.001. (E) Ordinary one way ANOVA with Sidak’s correction for multiple tests was performed. *p = 0.011, ***p < 0.001.

Article Snippet: Raw data and full Western Blot , Mendeley , https://doi.org/10.17632/7pfjtn7xhv.1.

Techniques: Concentration Assay, Activity Assay, Expressing, Phospho-proteomics, Western Blot, Immunoprecipitation, Two Tailed Test

Journal: Cell reports

Article Title: Steroid hormone catabolites activate the pyrin inflammasome through a non-canonical mechanism

doi: 10.1016/j.celrep.2022.111472

Figure Lengend Snippet:

Article Snippet: Raw data and full Western Blot , Mendeley , https://doi.org/10.17632/7pfjtn7xhv.1.

Techniques: Virus, Recombinant, Purification, Western Blot, Derivative Assay, Software

Journal: Nature Communications

Article Title: An oomycete plant pathogen reprograms host pre-mRNA splicing to subvert immunity

doi: 10.1038/s41467-017-02233-5

Figure Lengend Snippet: PsAvr3c physically interacts with soybean GmSKRP1/2 in vitro and in vivo. a PsAvr3c interacts with GmSKRP1/2 in yeast. PsAvr3c and GmSKRP1/2 were cloned into bait plasmid pGBKT7 (BD) and prey plasmid pGADT7 (AD), respectively. Yeast transformants were grown on SD/-Trp/-Leu (SD-2) and selected on SD/-Trp/-Leu/-His/-Ade (SD-4). The plates were photographed 5 days after inoculation. Experiments were repeated three times with similar results. b PsAvr3c physically interacts with GmSKRP1/2 in vitro. GST-GmSKRP1/2 or GST bound resins were incubated with E . coli supernatant containing His-PsAvr3c. Protein bands of GST-GmSKRP1/2 are marked by asterisks. The presence of His-tagged proteins was detected by western blot using anti-His tag antibody. c PsAvr3c interacts with GmSKRP1/2 in vivo. Co-immunoprecipitations (IP) were performed in extracts of N . benthamiana leaves expressing both FLAG-PsAvr3c and GFP-GmSKRP1/2. The presence of FLAG proteins was detected by western blot using anti-FLAG antibody. The GFP-GmSKRPs protein bands are indicated by asterisks and the protein loading is indicated by Ponceau stain. d Measuring binding affinity between PsAvr3c and GmSKRP1 in vitro. The binding affinity was determined using isothermal titration calorimetry (ITC). The raw titration data were integrated and fitted to a one-site binding model using the MicroCal PEAQ-ITC analysis software. Three original measurement results are 3.38, 3.52 and 3.14 μM, K D = 3.35 μM is mean value of three independent ITC experiments

Article Snippet: The raw titration data were integrated and fitted to a one-site binding model using the MicroCal PEAQ-ITC analysis software.

Techniques: In Vitro, In Vivo, Clone Assay, Plasmid Preparation, Incubation, Western Blot, Expressing, Staining, Binding Assay, Isothermal Titration Calorimetry, Titration, Software