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rabbit anti-gfp (chip)  (Thermo Fisher)


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    Thermo Fisher rabbit anti-gfp (chip)
    Rabbit Anti Gfp (Chip), supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rabbit+anti-gfp+(chip)/rabbit+anti+gfp/pm40239647-597-182-46
    Average 90 stars, based on 1 article reviews
    rabbit anti-gfp (chip) - by Bioz Stars, 2026-10
    90/100 stars

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    other:

    Article Title: Global regulation via modulation of ribosome pausing by the ABC-F protein EttA
    Article Snippet: Blinding was not relevant for this study, since the outcomes of the experiments are not dependent of the judgment of the researcher but depend of control reactions. re p o rtfo lio | rep o rtin g su m m ary A pril2023 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Validation Novel plant genotypes Seed stocks Authentication Plants Anti-GFP antibody : Thermo Fisher, Invitrogen GFP Antibody (A-11122) Secondary Antibody anti rabbit IRdye 800CW : Li-cor (926-32213) Secondary Antibody anti rabbit HRP : Promega, Anti-Rabbit IgG HRP Conjugate (W401B) We carried out Western blot on a protein lysates from strains that express or not the YFP protein .

    Article Title: Genetically-encoded markers for confocal visualization of single dense core vesicles
    Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Validation rabbit anti-GFP(Thermo Fisher,A-11122), rabbit anti-RFP(Takara,632453), mouse anti-GFP (Sigma-Aldrich, G1546), chicken anti-GFP (Invitrogen,A10262), mouse anti-PDF(DSHB, C7), mouse anti-Csp(DSHB, ab49), rabbit anti-CCAP(Jena Bioscience,ABD-033), rabbit anti-sNPF(Dr. Jan Veenstra), mouse anti-pBurs (Aaron Hsueh).

    Article Title: GluN2A mediates ketamine-induced rapid antidepressant-like responses.
    Article Snippet: Ketamine was thought to induce rapid antidepressant responses by inhibiting GluN2B-containing N-methyl-d-aspartic acid (NMDA) receptors (NMDARs), which presents a promising opportunity to develop better antidepressants.. However, adverse side effects limit the broader application of ketamine and GluN2B inhibitors are yet to be approved for clinical use.. It is unclear whether ketamine acts solely through GluN2B-dependent mechanisms.

    Article Title: A systematic analysis of Trypanosoma brucei chromatin factors identifies novel protein interaction networks associated with sites of transcription initiation and termination
    Article Snippet: Rabbit anti-GFP primary antibody (Thermo Fisher Scientific A-11122) was used at 1:500 dilution, and secondary Alexa fluor 568 goat antirabbit antibody (Thermo Fisher Scientific A-11036) was used at 1:1000 dilution.

    Article Title: HLTF Prevents G4 Accumulation and Promotes G4-induced Fork Slowing to Maintain Genome Stability
    Article Snippet: The primary antibodies were diluted in 3% BSA/PBS and incubated overnight at 4°C: rabbit anti-G4 (clone 1H6, Absolute Antibody ab00389-23.0, 1:500), goat anti-G4 (clone 1H6, Absolute Antibody ab00389-24.1, 1:500), anti-G4 (clone BG4, Sigma-Aldrich MABE917, 1:500), rabbit anti-GFP (Abcam ab290, 1:1000), rabbit anti-GFP (Thermo Fisher Scientific A-11122, 1:1000), rabbit anti-HLTF (Abcam ab183042, 1:1000), mouse anti-MSH2 (Abcam ab52266, 1:1000), mouse anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) (Abcam ab24758, 1:500), rabbit anti-RPA34 (Abcam ab97594, 1:1000).

    Article Title: Neural circuit mechanisms underlying context-specific halting in Drosophila.
    Article Snippet: Alexa fluor secondary antibodies (Thermo Fisher Scientific) were used at 1:500 dilution (Goat anti-chicken: Alexa488, AB_2576217, Goat anti-rabbit: Alex568, AB_10563566, Goat anti-mouse: Alex568, AB_2534072 and Goat anti-mouse: Alex647, AB_141725).

    Virus:

    Article Title: Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice.
    Article Snippet: Article Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice

    Recombinant:

    Article Title: Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice.
    Article Snippet: Article Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice

    Protease Inhibitor:

    Article Title: Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice.
    Article Snippet: Article Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice

    Reporter Assay:

    Article Title: Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice.
    Article Snippet: Article Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice

    Cloning:

    Article Title: Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice.
    Article Snippet: Article Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice

    Quantitative RT-PCR:

    Article Title: Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice.
    Article Snippet: Article Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice

    ChIP-qPCR:

    Article Title: Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice.
    Article Snippet: Article Linking glucose signaling to nitrogen utilization by the OsHXK7-ARE4 complex in rice



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    Santa Cruz Biotechnology resource source identifier antibodies rabbit polyclonal anti p65 santa cruz sc 372 chip seq
    Figure 2. LPS-Induced LSD1 Phosphorylation Is Required for the Activation of NF-kB Target Genes at the Genome-wide Level (A) Flow chart showing the strategy of RNA-seq analysis. (B) Hierarchical clustering results applied to 3,558 differentially expressed genes (DEGs). The <t>p65</t> peaks around the TSS (±2.5 kbps) were shown. (C) GO analysis for the genes in cluster 1 showing that cytokine production and inflammatory response genes are significantly enriched in cluster 1. (D) De novo motif analysis for the p65 peaks close to the TSSs of the genes in cluster 1. Hypergeometric p values were calculated. (E) qRT-PCR analysis of LSD1 phosphorylation-dependent target genes was conducted using WT or Lsd1SA/SA BMDMs untreated or treated with LPS for 2 hr. (F) qRT-PCR analysis of LSD1 phosphorylation-dependent target genes was conducted using lung tissue extracts obtained from vehicle or LPS intraperitoneally injected WT or Lsd1SA/SA mice for 6 hr (n = 6 per group). (G) qRT-PCR analysis of LSD1 phosphorylation-dependent target genes was conducted using lungs from WT mice (n = 6 per group). Mice were intraperitoneally injected with Go6976 (1 mg/kg body weight) or equal volume of vehicle for 1 hr prior to LPS challenge (10 mg/kg body weight) for 6 hr. Data are expressed as mean ± SD; n = 3. *p < 0.05, ***p < 0.001 (two-way ANOVA). See also Figures S2 and S3 and Tables S1, S2, and S3.
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    Thermo Fisher chip grade rabbit anti gfp
    WipB is targeted to lysosomes by its C-terminal domain where it interacts with components of the lysosomal nutrient sensing system ( a ) HeLa cells expressing <t>GFP-fusion</t> proteins of WipB, WipB D118A , WipB 1-364 and WipB 365-524 . Scale bar, 10 µm. ( b ) HeLa cells expressing GFP-WipB (green) stained using an anti-LAMP-1 antibody (Magenta) after permeabilisation and fixation. Scale bar, 10 µm. ( c ) HeLa cells expressing GFP-WipB (green) were incubated with Lysotracker (red) for 15 min before fixation. Scale bar, 10 µm. ( d ) SDS-PAGE of fractions following co-immunoprecipitation of GFP, GFP-WipB or the indicated GFP-WipB derivatives from HeLa cell lysates <t>using</t> <t>anti-GFP</t> antibody and immunoblotting with anti-v-ATPase A, -v-ATPase B or LAMTOR1 antibodies. A cropped blot is here displayed and the corresponding full-length blot is included in the supplementary information. ( e ) SDS-PAGE of fractions following co-immunoprecipitation of v-ATPase A, v-ATPase B or LAMTOR1 from lysates of HeLa cells expressing GFP or GFP-WipB and immunoblotting <t>with</t> <t>anti-GFP</t> antibody. A cropped blot is here displayed and the corresponding full-length blot is included in the supplementary information.
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    Figure 2. LPS-Induced LSD1 Phosphorylation Is Required for the Activation of NF-kB Target Genes at the Genome-wide Level (A) Flow chart showing the strategy of RNA-seq analysis. (B) Hierarchical clustering results applied to 3,558 differentially expressed genes (DEGs). The p65 peaks around the TSS (±2.5 kbps) were shown. (C) GO analysis for the genes in cluster 1 showing that cytokine production and inflammatory response genes are significantly enriched in cluster 1. (D) De novo motif analysis for the p65 peaks close to the TSSs of the genes in cluster 1. Hypergeometric p values were calculated. (E) qRT-PCR analysis of LSD1 phosphorylation-dependent target genes was conducted using WT or Lsd1SA/SA BMDMs untreated or treated with LPS for 2 hr. (F) qRT-PCR analysis of LSD1 phosphorylation-dependent target genes was conducted using lung tissue extracts obtained from vehicle or LPS intraperitoneally injected WT or Lsd1SA/SA mice for 6 hr (n = 6 per group). (G) qRT-PCR analysis of LSD1 phosphorylation-dependent target genes was conducted using lungs from WT mice (n = 6 per group). Mice were intraperitoneally injected with Go6976 (1 mg/kg body weight) or equal volume of vehicle for 1 hr prior to LPS challenge (10 mg/kg body weight) for 6 hr. Data are expressed as mean ± SD; n = 3. *p < 0.05, ***p < 0.001 (two-way ANOVA). See also Figures S2 and S3 and Tables S1, S2, and S3.

    Journal: Molecular cell

    Article Title: PKCα-LSD1-NF-κB-Signaling Cascade Is Crucial for Epigenetic Control of the Inflammatory Response.

    doi: 10.1016/j.molcel.2018.01.002

    Figure Lengend Snippet: Figure 2. LPS-Induced LSD1 Phosphorylation Is Required for the Activation of NF-kB Target Genes at the Genome-wide Level (A) Flow chart showing the strategy of RNA-seq analysis. (B) Hierarchical clustering results applied to 3,558 differentially expressed genes (DEGs). The p65 peaks around the TSS (±2.5 kbps) were shown. (C) GO analysis for the genes in cluster 1 showing that cytokine production and inflammatory response genes are significantly enriched in cluster 1. (D) De novo motif analysis for the p65 peaks close to the TSSs of the genes in cluster 1. Hypergeometric p values were calculated. (E) qRT-PCR analysis of LSD1 phosphorylation-dependent target genes was conducted using WT or Lsd1SA/SA BMDMs untreated or treated with LPS for 2 hr. (F) qRT-PCR analysis of LSD1 phosphorylation-dependent target genes was conducted using lung tissue extracts obtained from vehicle or LPS intraperitoneally injected WT or Lsd1SA/SA mice for 6 hr (n = 6 per group). (G) qRT-PCR analysis of LSD1 phosphorylation-dependent target genes was conducted using lungs from WT mice (n = 6 per group). Mice were intraperitoneally injected with Go6976 (1 mg/kg body weight) or equal volume of vehicle for 1 hr prior to LPS challenge (10 mg/kg body weight) for 6 hr. Data are expressed as mean ± SD; n = 3. *p < 0.05, ***p < 0.001 (two-way ANOVA). See also Figures S2 and S3 and Tables S1, S2, and S3.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-p65 Santa Cruz sc-372 (ChIP-seq) ; RRID: AB_632037 Goat polyclonal anti-Lamin A/C Santa Cruz sc-6215 ; RRID: AB_648152 Rabbit polyclonal anti-GST Santa Cruz sc-459 ; RRID: AB_631586 Mouse monoclonal anti-GFP Santa Cruz sc-9996 ; RRID: AB_627695 Rabbit polyclonal anti-LSD1 Cell Signaling #2139 ; RRID: AB_2070135 Rabbit polyclonal anti-PKCa Cell Signaling #2056S ; RRID: AB_2284227 Rabbit polyclonal anti-p65 abcam ab7970 ; RRID: AB_306184 Rabbit polyclonal anti-LSD1 abcam ab17721 (ChIP-seq) ; RRID: AB_443964 Rabbit polyclonal anti-H3K9Ac abcam ab4441 ; RRID: AB_2118292 Rabbit polyclonal anti-H3K9me2 abcam ab1220 ; RRID: AB_449854 Rabbit polyclonal anti-H3K4me2 abcam ab32356 ; RRID: AB_732924 Rabbit polyclonal anti-LSD1 NOVUS NB-100-1762 ; RRID: AB_10001568 Rabbit polyclonal anti-PKCa NOVUS NB-110-57356 ; RRID: AB_844168 Rabbit polyclonal anti-C/EBPd NOVUS NB-110-85519 ; RRID: AB_1201191 Rabbit polyclonal anti-phospho(S657)-PKCa Millipore #06-822 ; RRID: AB_11213245 Rabbit polyclonal anti-phospho(S112)-LSD1 Millipore ABE 1462 ; RRID: N/A Mouse monoclonal anti-HA Covance MMS-101R ; RRID: AB_291263 Mouse monoclonal anti-b-actin Sigma A1978 ; RRID: AB_476692 Rabbit polyclonal anti-Tubulin AbFrontier LF-PA0146A ; RRID: N/A Rabbit polyclonal anti-methyl(K314/315)-p65 Elabscience biotechnology ENH006 ; RRID: N/A Mouse monoclonal anti-FK2 Enzo Life Sciences BML-PW8810 ; RRID: AB_10541840 Bacterial and Virus Strains DH5-a Escherichia coli Enzynomics CP010 Rossetta Escherichia coli Novagen 70954-3CN M15 pREP4 Escherichia coli QIAGEN 32149 Chemicals, Peptides, and Recombinant Proteins Lipopolysaccharides (LPS) from Escherichia coli Sigma L3129 GSK-LSD1 Sigma SML1072 Go6976 Cayman 13310 MG132 A.G. Scientific M-1157 DMSO (Vehicle) Sigma D2650 Ni-NTA Agarose QIAGEN 30210 cOmplete Protease Inhibitor Cocktail Roche 11697498001 TNT T7 Quick Coupled Transcription/Translation System Promega L1170 l-phosphatase NEB P0753 Human p65 protein (TNT) This paper N/A Human GST-LSD1 protein (GST purification) This paper N/A Human GST-p65 protein (GST purification) This paper N/A Human GST-SET7/9 protein (GST purification) This paper N/A Critical Commercial Assays TruSeq RNA Sample prep kit v2 Illumina RS-122-2001 Illumina Hi-seq 3000/4000 SBS kit v3 Illumina FC-410-1003 (Continued on next page) e1 Molecular Cell 69, 398–411.e1–e6, February 1, 2018

    Techniques: Phospho-proteomics, Activation Assay, Genome Wide, RNA Sequencing, Quantitative RT-PCR, Injection

    Figure 3. LPS-Induced LSD1 Phosphorylation Is Required for the Recruitment of p65 to Target Promoters (A) Co-immunoprecipitation assay of p65 with LSD1 was conducted in nuclear fraction of BMDMs untreated or treated with LPS for 2 hr. (B) GST-LSD1 and GST-p65 proteins purified from E. coli were used as substrates for in vitro kinase assay using WT or dominant-negative mutant (DN) of PKCa.

    Journal: Molecular cell

    Article Title: PKCα-LSD1-NF-κB-Signaling Cascade Is Crucial for Epigenetic Control of the Inflammatory Response.

    doi: 10.1016/j.molcel.2018.01.002

    Figure Lengend Snippet: Figure 3. LPS-Induced LSD1 Phosphorylation Is Required for the Recruitment of p65 to Target Promoters (A) Co-immunoprecipitation assay of p65 with LSD1 was conducted in nuclear fraction of BMDMs untreated or treated with LPS for 2 hr. (B) GST-LSD1 and GST-p65 proteins purified from E. coli were used as substrates for in vitro kinase assay using WT or dominant-negative mutant (DN) of PKCa.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-p65 Santa Cruz sc-372 (ChIP-seq) ; RRID: AB_632037 Goat polyclonal anti-Lamin A/C Santa Cruz sc-6215 ; RRID: AB_648152 Rabbit polyclonal anti-GST Santa Cruz sc-459 ; RRID: AB_631586 Mouse monoclonal anti-GFP Santa Cruz sc-9996 ; RRID: AB_627695 Rabbit polyclonal anti-LSD1 Cell Signaling #2139 ; RRID: AB_2070135 Rabbit polyclonal anti-PKCa Cell Signaling #2056S ; RRID: AB_2284227 Rabbit polyclonal anti-p65 abcam ab7970 ; RRID: AB_306184 Rabbit polyclonal anti-LSD1 abcam ab17721 (ChIP-seq) ; RRID: AB_443964 Rabbit polyclonal anti-H3K9Ac abcam ab4441 ; RRID: AB_2118292 Rabbit polyclonal anti-H3K9me2 abcam ab1220 ; RRID: AB_449854 Rabbit polyclonal anti-H3K4me2 abcam ab32356 ; RRID: AB_732924 Rabbit polyclonal anti-LSD1 NOVUS NB-100-1762 ; RRID: AB_10001568 Rabbit polyclonal anti-PKCa NOVUS NB-110-57356 ; RRID: AB_844168 Rabbit polyclonal anti-C/EBPd NOVUS NB-110-85519 ; RRID: AB_1201191 Rabbit polyclonal anti-phospho(S657)-PKCa Millipore #06-822 ; RRID: AB_11213245 Rabbit polyclonal anti-phospho(S112)-LSD1 Millipore ABE 1462 ; RRID: N/A Mouse monoclonal anti-HA Covance MMS-101R ; RRID: AB_291263 Mouse monoclonal anti-b-actin Sigma A1978 ; RRID: AB_476692 Rabbit polyclonal anti-Tubulin AbFrontier LF-PA0146A ; RRID: N/A Rabbit polyclonal anti-methyl(K314/315)-p65 Elabscience biotechnology ENH006 ; RRID: N/A Mouse monoclonal anti-FK2 Enzo Life Sciences BML-PW8810 ; RRID: AB_10541840 Bacterial and Virus Strains DH5-a Escherichia coli Enzynomics CP010 Rossetta Escherichia coli Novagen 70954-3CN M15 pREP4 Escherichia coli QIAGEN 32149 Chemicals, Peptides, and Recombinant Proteins Lipopolysaccharides (LPS) from Escherichia coli Sigma L3129 GSK-LSD1 Sigma SML1072 Go6976 Cayman 13310 MG132 A.G. Scientific M-1157 DMSO (Vehicle) Sigma D2650 Ni-NTA Agarose QIAGEN 30210 cOmplete Protease Inhibitor Cocktail Roche 11697498001 TNT T7 Quick Coupled Transcription/Translation System Promega L1170 l-phosphatase NEB P0753 Human p65 protein (TNT) This paper N/A Human GST-LSD1 protein (GST purification) This paper N/A Human GST-p65 protein (GST purification) This paper N/A Human GST-SET7/9 protein (GST purification) This paper N/A Critical Commercial Assays TruSeq RNA Sample prep kit v2 Illumina RS-122-2001 Illumina Hi-seq 3000/4000 SBS kit v3 Illumina FC-410-1003 (Continued on next page) e1 Molecular Cell 69, 398–411.e1–e6, February 1, 2018

    Techniques: Phospho-proteomics, Co-Immunoprecipitation Assay, In Vitro, Kinase Assay, Dominant Negative Mutation

    Figure 4. Demethylation of p65 by LSD1 En- hances Protein Stability of p65 (A) Co-immunoprecipitation assay of p65 with WT, SA, or KA mutant of LSD1 in nuclear fraction of Raw264.7 cells transfected with WT, SA, or KA mutant of FLAG-tagged LSD1 upon MG132 pre- treatment for 4 hr and then LPS treatment for 2 hr. (B) In vivo demethylation assay was performed in nuclear fraction of Raw264.7 cells upon MG132 pre- treatment for 4 hr and then LPS treatment for 2 hr. After co-immunoprecipitation assay using anti-p65 antibody, the methylated p65 was detected by anti- methyl p65 antibody. (C) In vitro demethylation assay, using FLAG-p65 proteins purified from cell lysates as a substrate, GST-SET7/9 proteins purified from E. coli as a methyltransferase, and His-LSD1 proteins purified from E. coli as a demethylase, was performed. In vitro kinase assay using PKCa was conducted to obtain phosphorylated LSD1 before demethylation assay. The reaction mixtures were subject to SDS- PAGE analysis, and methylated p65 was detected by anti-methyl p65 antibody at K314/315 sites. (D) Immunoblot analysis was performed in nuclear fraction of WT and Lsd1SA/SA BMDMs with LPS treatment for 2 hr in the absence or presence of MG132 pre-treatment for 4 hr using the indicated antibodies. (E) Protein extracts from Raw264.7 cells transfected with WT, SA, or KA mutant of FLAG-tagged LSD1 upon MG132 pre-treatment for 4 hr and then LPS treatment for 2 hr were subjected to pull-down with Ni2+-NTA beads. Ubiquitination of p65 was assessed by anti-p65 antibody. (F) Co-immunoprecipitation assay using anti-p65 antibody in Raw264.7 cells with MG132 pre-treat- ment for 4 hr and then LPS treatment for 2 hr in the absence or presence of Go6976 or GSK-LSD1 for 6 hr. Ubiquitination of p65 was assessed by anti-FK2 antibody. (G) Schematic representation of LSD1 phosphory- lation-mediated p65 demethylation resulting in the p65 stabilization. See also Figure S5.

    Journal: Molecular cell

    Article Title: PKCα-LSD1-NF-κB-Signaling Cascade Is Crucial for Epigenetic Control of the Inflammatory Response.

    doi: 10.1016/j.molcel.2018.01.002

    Figure Lengend Snippet: Figure 4. Demethylation of p65 by LSD1 En- hances Protein Stability of p65 (A) Co-immunoprecipitation assay of p65 with WT, SA, or KA mutant of LSD1 in nuclear fraction of Raw264.7 cells transfected with WT, SA, or KA mutant of FLAG-tagged LSD1 upon MG132 pre- treatment for 4 hr and then LPS treatment for 2 hr. (B) In vivo demethylation assay was performed in nuclear fraction of Raw264.7 cells upon MG132 pre- treatment for 4 hr and then LPS treatment for 2 hr. After co-immunoprecipitation assay using anti-p65 antibody, the methylated p65 was detected by anti- methyl p65 antibody. (C) In vitro demethylation assay, using FLAG-p65 proteins purified from cell lysates as a substrate, GST-SET7/9 proteins purified from E. coli as a methyltransferase, and His-LSD1 proteins purified from E. coli as a demethylase, was performed. In vitro kinase assay using PKCa was conducted to obtain phosphorylated LSD1 before demethylation assay. The reaction mixtures were subject to SDS- PAGE analysis, and methylated p65 was detected by anti-methyl p65 antibody at K314/315 sites. (D) Immunoblot analysis was performed in nuclear fraction of WT and Lsd1SA/SA BMDMs with LPS treatment for 2 hr in the absence or presence of MG132 pre-treatment for 4 hr using the indicated antibodies. (E) Protein extracts from Raw264.7 cells transfected with WT, SA, or KA mutant of FLAG-tagged LSD1 upon MG132 pre-treatment for 4 hr and then LPS treatment for 2 hr were subjected to pull-down with Ni2+-NTA beads. Ubiquitination of p65 was assessed by anti-p65 antibody. (F) Co-immunoprecipitation assay using anti-p65 antibody in Raw264.7 cells with MG132 pre-treat- ment for 4 hr and then LPS treatment for 2 hr in the absence or presence of Go6976 or GSK-LSD1 for 6 hr. Ubiquitination of p65 was assessed by anti-FK2 antibody. (G) Schematic representation of LSD1 phosphory- lation-mediated p65 demethylation resulting in the p65 stabilization. See also Figure S5.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-p65 Santa Cruz sc-372 (ChIP-seq) ; RRID: AB_632037 Goat polyclonal anti-Lamin A/C Santa Cruz sc-6215 ; RRID: AB_648152 Rabbit polyclonal anti-GST Santa Cruz sc-459 ; RRID: AB_631586 Mouse monoclonal anti-GFP Santa Cruz sc-9996 ; RRID: AB_627695 Rabbit polyclonal anti-LSD1 Cell Signaling #2139 ; RRID: AB_2070135 Rabbit polyclonal anti-PKCa Cell Signaling #2056S ; RRID: AB_2284227 Rabbit polyclonal anti-p65 abcam ab7970 ; RRID: AB_306184 Rabbit polyclonal anti-LSD1 abcam ab17721 (ChIP-seq) ; RRID: AB_443964 Rabbit polyclonal anti-H3K9Ac abcam ab4441 ; RRID: AB_2118292 Rabbit polyclonal anti-H3K9me2 abcam ab1220 ; RRID: AB_449854 Rabbit polyclonal anti-H3K4me2 abcam ab32356 ; RRID: AB_732924 Rabbit polyclonal anti-LSD1 NOVUS NB-100-1762 ; RRID: AB_10001568 Rabbit polyclonal anti-PKCa NOVUS NB-110-57356 ; RRID: AB_844168 Rabbit polyclonal anti-C/EBPd NOVUS NB-110-85519 ; RRID: AB_1201191 Rabbit polyclonal anti-phospho(S657)-PKCa Millipore #06-822 ; RRID: AB_11213245 Rabbit polyclonal anti-phospho(S112)-LSD1 Millipore ABE 1462 ; RRID: N/A Mouse monoclonal anti-HA Covance MMS-101R ; RRID: AB_291263 Mouse monoclonal anti-b-actin Sigma A1978 ; RRID: AB_476692 Rabbit polyclonal anti-Tubulin AbFrontier LF-PA0146A ; RRID: N/A Rabbit polyclonal anti-methyl(K314/315)-p65 Elabscience biotechnology ENH006 ; RRID: N/A Mouse monoclonal anti-FK2 Enzo Life Sciences BML-PW8810 ; RRID: AB_10541840 Bacterial and Virus Strains DH5-a Escherichia coli Enzynomics CP010 Rossetta Escherichia coli Novagen 70954-3CN M15 pREP4 Escherichia coli QIAGEN 32149 Chemicals, Peptides, and Recombinant Proteins Lipopolysaccharides (LPS) from Escherichia coli Sigma L3129 GSK-LSD1 Sigma SML1072 Go6976 Cayman 13310 MG132 A.G. Scientific M-1157 DMSO (Vehicle) Sigma D2650 Ni-NTA Agarose QIAGEN 30210 cOmplete Protease Inhibitor Cocktail Roche 11697498001 TNT T7 Quick Coupled Transcription/Translation System Promega L1170 l-phosphatase NEB P0753 Human p65 protein (TNT) This paper N/A Human GST-LSD1 protein (GST purification) This paper N/A Human GST-p65 protein (GST purification) This paper N/A Human GST-SET7/9 protein (GST purification) This paper N/A Critical Commercial Assays TruSeq RNA Sample prep kit v2 Illumina RS-122-2001 Illumina Hi-seq 3000/4000 SBS kit v3 Illumina FC-410-1003 (Continued on next page) e1 Molecular Cell 69, 398–411.e1–e6, February 1, 2018

    Techniques: Co-Immunoprecipitation Assay, Mutagenesis, Transfection, In Vivo, Demethylation Assay, Methylation, In Vitro, Kinase Assay, SDS Page, Western Blot, Ubiquitin Proteomics

    Figure 5. PKCa-LSD1 Phosphorylation Axis Controls the Response to Prolonged Inflammation (A) qRT-PCR analysis of Mcp-1, Il-6, and Cebpd mRNAs in WT and Lsd1SA/SA BMDMs with LPS treatment for the indicated times. (B) qRT-PCR analysis of Mcp-1, Il-6, and Cebpd mRNAs in WT BMDMs with LPS treatment for the indicated times in the absence or presence of Go6976 pre- treatment for 6 hr. (C) The binding profile of endogenous p65 to LSD1 in nuclear fraction of BMDMs with LPS treatment over an indicated time course.

    Journal: Molecular cell

    Article Title: PKCα-LSD1-NF-κB-Signaling Cascade Is Crucial for Epigenetic Control of the Inflammatory Response.

    doi: 10.1016/j.molcel.2018.01.002

    Figure Lengend Snippet: Figure 5. PKCa-LSD1 Phosphorylation Axis Controls the Response to Prolonged Inflammation (A) qRT-PCR analysis of Mcp-1, Il-6, and Cebpd mRNAs in WT and Lsd1SA/SA BMDMs with LPS treatment for the indicated times. (B) qRT-PCR analysis of Mcp-1, Il-6, and Cebpd mRNAs in WT BMDMs with LPS treatment for the indicated times in the absence or presence of Go6976 pre- treatment for 6 hr. (C) The binding profile of endogenous p65 to LSD1 in nuclear fraction of BMDMs with LPS treatment over an indicated time course.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-p65 Santa Cruz sc-372 (ChIP-seq) ; RRID: AB_632037 Goat polyclonal anti-Lamin A/C Santa Cruz sc-6215 ; RRID: AB_648152 Rabbit polyclonal anti-GST Santa Cruz sc-459 ; RRID: AB_631586 Mouse monoclonal anti-GFP Santa Cruz sc-9996 ; RRID: AB_627695 Rabbit polyclonal anti-LSD1 Cell Signaling #2139 ; RRID: AB_2070135 Rabbit polyclonal anti-PKCa Cell Signaling #2056S ; RRID: AB_2284227 Rabbit polyclonal anti-p65 abcam ab7970 ; RRID: AB_306184 Rabbit polyclonal anti-LSD1 abcam ab17721 (ChIP-seq) ; RRID: AB_443964 Rabbit polyclonal anti-H3K9Ac abcam ab4441 ; RRID: AB_2118292 Rabbit polyclonal anti-H3K9me2 abcam ab1220 ; RRID: AB_449854 Rabbit polyclonal anti-H3K4me2 abcam ab32356 ; RRID: AB_732924 Rabbit polyclonal anti-LSD1 NOVUS NB-100-1762 ; RRID: AB_10001568 Rabbit polyclonal anti-PKCa NOVUS NB-110-57356 ; RRID: AB_844168 Rabbit polyclonal anti-C/EBPd NOVUS NB-110-85519 ; RRID: AB_1201191 Rabbit polyclonal anti-phospho(S657)-PKCa Millipore #06-822 ; RRID: AB_11213245 Rabbit polyclonal anti-phospho(S112)-LSD1 Millipore ABE 1462 ; RRID: N/A Mouse monoclonal anti-HA Covance MMS-101R ; RRID: AB_291263 Mouse monoclonal anti-b-actin Sigma A1978 ; RRID: AB_476692 Rabbit polyclonal anti-Tubulin AbFrontier LF-PA0146A ; RRID: N/A Rabbit polyclonal anti-methyl(K314/315)-p65 Elabscience biotechnology ENH006 ; RRID: N/A Mouse monoclonal anti-FK2 Enzo Life Sciences BML-PW8810 ; RRID: AB_10541840 Bacterial and Virus Strains DH5-a Escherichia coli Enzynomics CP010 Rossetta Escherichia coli Novagen 70954-3CN M15 pREP4 Escherichia coli QIAGEN 32149 Chemicals, Peptides, and Recombinant Proteins Lipopolysaccharides (LPS) from Escherichia coli Sigma L3129 GSK-LSD1 Sigma SML1072 Go6976 Cayman 13310 MG132 A.G. Scientific M-1157 DMSO (Vehicle) Sigma D2650 Ni-NTA Agarose QIAGEN 30210 cOmplete Protease Inhibitor Cocktail Roche 11697498001 TNT T7 Quick Coupled Transcription/Translation System Promega L1170 l-phosphatase NEB P0753 Human p65 protein (TNT) This paper N/A Human GST-LSD1 protein (GST purification) This paper N/A Human GST-p65 protein (GST purification) This paper N/A Human GST-SET7/9 protein (GST purification) This paper N/A Critical Commercial Assays TruSeq RNA Sample prep kit v2 Illumina RS-122-2001 Illumina Hi-seq 3000/4000 SBS kit v3 Illumina FC-410-1003 (Continued on next page) e1 Molecular Cell 69, 398–411.e1–e6, February 1, 2018

    Techniques: Phospho-proteomics, Quantitative RT-PCR, Binding Assay

    Figure 6. Sustained Expression of p65 in the Nucleus and Subsequent Activation of Inflammation Depends on the PKCa-LSD1- NF-kB-Signaling Cascade (A) Immunoblot analysis was performed in nuclear fraction of WT and Lsd1SA/SA BMDMs with LPS treatment for the indicated times using the indicated antibodies. (B) Representative confocal images of immunocy- tochemistry. Immunocytochemistry was performed in Lsd1/ MEFs reconstituted with WT, SA, or KA mutant of HA-tagged LSD1 treated with LPS for the indicated times. HA, green; p65, red; DAPI, blue. Scale bar, 20 mm. (C and D) Immunoblot analysis was performed in nuclear fraction of BMDMs (C) and Raw264.7 cells (D) using the indicated antibodies. Pre-treatment with Go6976 or GSK-LSD1 for 6 hr was followed by LPS treatment for the indicated times before collection. (E) Immunoblot analysis was performed in nuclear fraction of Raw264.7 cells using the indicated anti- bodies. Go6976 or GSK-LSD1 is pre-treated for 6 hr followed by LPS treatment for 2 hr in the absence or presence of MG132 for 4 hr.

    Journal: Molecular cell

    Article Title: PKCα-LSD1-NF-κB-Signaling Cascade Is Crucial for Epigenetic Control of the Inflammatory Response.

    doi: 10.1016/j.molcel.2018.01.002

    Figure Lengend Snippet: Figure 6. Sustained Expression of p65 in the Nucleus and Subsequent Activation of Inflammation Depends on the PKCa-LSD1- NF-kB-Signaling Cascade (A) Immunoblot analysis was performed in nuclear fraction of WT and Lsd1SA/SA BMDMs with LPS treatment for the indicated times using the indicated antibodies. (B) Representative confocal images of immunocy- tochemistry. Immunocytochemistry was performed in Lsd1/ MEFs reconstituted with WT, SA, or KA mutant of HA-tagged LSD1 treated with LPS for the indicated times. HA, green; p65, red; DAPI, blue. Scale bar, 20 mm. (C and D) Immunoblot analysis was performed in nuclear fraction of BMDMs (C) and Raw264.7 cells (D) using the indicated antibodies. Pre-treatment with Go6976 or GSK-LSD1 for 6 hr was followed by LPS treatment for the indicated times before collection. (E) Immunoblot analysis was performed in nuclear fraction of Raw264.7 cells using the indicated anti- bodies. Go6976 or GSK-LSD1 is pre-treated for 6 hr followed by LPS treatment for 2 hr in the absence or presence of MG132 for 4 hr.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-p65 Santa Cruz sc-372 (ChIP-seq) ; RRID: AB_632037 Goat polyclonal anti-Lamin A/C Santa Cruz sc-6215 ; RRID: AB_648152 Rabbit polyclonal anti-GST Santa Cruz sc-459 ; RRID: AB_631586 Mouse monoclonal anti-GFP Santa Cruz sc-9996 ; RRID: AB_627695 Rabbit polyclonal anti-LSD1 Cell Signaling #2139 ; RRID: AB_2070135 Rabbit polyclonal anti-PKCa Cell Signaling #2056S ; RRID: AB_2284227 Rabbit polyclonal anti-p65 abcam ab7970 ; RRID: AB_306184 Rabbit polyclonal anti-LSD1 abcam ab17721 (ChIP-seq) ; RRID: AB_443964 Rabbit polyclonal anti-H3K9Ac abcam ab4441 ; RRID: AB_2118292 Rabbit polyclonal anti-H3K9me2 abcam ab1220 ; RRID: AB_449854 Rabbit polyclonal anti-H3K4me2 abcam ab32356 ; RRID: AB_732924 Rabbit polyclonal anti-LSD1 NOVUS NB-100-1762 ; RRID: AB_10001568 Rabbit polyclonal anti-PKCa NOVUS NB-110-57356 ; RRID: AB_844168 Rabbit polyclonal anti-C/EBPd NOVUS NB-110-85519 ; RRID: AB_1201191 Rabbit polyclonal anti-phospho(S657)-PKCa Millipore #06-822 ; RRID: AB_11213245 Rabbit polyclonal anti-phospho(S112)-LSD1 Millipore ABE 1462 ; RRID: N/A Mouse monoclonal anti-HA Covance MMS-101R ; RRID: AB_291263 Mouse monoclonal anti-b-actin Sigma A1978 ; RRID: AB_476692 Rabbit polyclonal anti-Tubulin AbFrontier LF-PA0146A ; RRID: N/A Rabbit polyclonal anti-methyl(K314/315)-p65 Elabscience biotechnology ENH006 ; RRID: N/A Mouse monoclonal anti-FK2 Enzo Life Sciences BML-PW8810 ; RRID: AB_10541840 Bacterial and Virus Strains DH5-a Escherichia coli Enzynomics CP010 Rossetta Escherichia coli Novagen 70954-3CN M15 pREP4 Escherichia coli QIAGEN 32149 Chemicals, Peptides, and Recombinant Proteins Lipopolysaccharides (LPS) from Escherichia coli Sigma L3129 GSK-LSD1 Sigma SML1072 Go6976 Cayman 13310 MG132 A.G. Scientific M-1157 DMSO (Vehicle) Sigma D2650 Ni-NTA Agarose QIAGEN 30210 cOmplete Protease Inhibitor Cocktail Roche 11697498001 TNT T7 Quick Coupled Transcription/Translation System Promega L1170 l-phosphatase NEB P0753 Human p65 protein (TNT) This paper N/A Human GST-LSD1 protein (GST purification) This paper N/A Human GST-p65 protein (GST purification) This paper N/A Human GST-SET7/9 protein (GST purification) This paper N/A Critical Commercial Assays TruSeq RNA Sample prep kit v2 Illumina RS-122-2001 Illumina Hi-seq 3000/4000 SBS kit v3 Illumina FC-410-1003 (Continued on next page) e1 Molecular Cell 69, 398–411.e1–e6, February 1, 2018

    Techniques: Expressing, Activation Assay, Western Blot, Immunocytochemistry, Mutagenesis

    Figure 7. Inhibition of PKCa Activity or LSD1 Activity in Mice Shows Attenuated Septic- Induced Mortality (A) Immunoblot analysis of lungs from WT mice (n = 3 per group). Mice were intraperitoneally injected with Go6976 (1 mg/kg body weight), GSK- LSD1 (1 mg/kg body weight), or an equal volume of vehicle for 1 hr prior to LPS challenge (10 mg/kg body weight) for 6 hr. (B) WT (blue line) or Lsd1SA/SA (red line) mice were subjected to CLP (n = 20 each). Animal survival was monitored every 6 hr for 144 hr after CLP. (C and D) WT mice (n = 20) were administered with Go6976 (green line) or an equal volume of vehicle (blue line) (C) or GSK-LSD1 (purple line) or an equal volume of vehicle (blue line) (D) after CLP (1 mg/kg body weight, each, intravenous [i.v.], at 12 and 50 hr after CLP). Animal survival was monitored every 6 hr for 144 hr after CLP. **p < 0.01 (log-rank test) (B–D). (E) Representative images of H&E staining of lung sections from WT and Lsd1SA/SA mice after CLP (n = 6 per group) or from WT mice intravenously in- jected with Go6976, GSK-LSD1, or vehicle (n = 6 per group, 1 mg/kg body weight, at 12 and 50 hr after CLP). Mice were euthanized 72 hr after CLP. Scale bars, 200 mm. (F and G) Plasma concentrations of cytokines (MCP-1, IL-6, and TNF-a) were measured 72 hr after CLP in WT and Lsd1SA/SA mice (F) or in WT mice intravenously injected (1 mg/kg body weight) with Go6976 or GSK-LSD1 at 12 and 50 hr after CLP (G). (H and I) Septic injury marker (ALT, LDH, and BUN) levels were measured 72 hr after CLP in WT and Lsd1SA/SA mice (H) or in WT mice intravenously in- jected (1 mg/kg body weight) with Go6976 or GSK- LSD1 at 12 and 50 hr after CLP (I). Data are expressed as mean ± SD; n = 5; *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA) (F and H). Data are expressed as mean ± SD; n = 5 per group; *p < 0.05, **p < 0.01 (unpaired two-tailed Student’s t test) (G and I). (J) Schematic model of the PKCa-LSD1-NF-kB- signaling axis showing that blocking PKCa or LSD1 activity decreases p65 protein stability and, thus, induces resistance to acute systemic inflammation, leading to increased survival rates of sepsis in Lsd1SA/SA mice compared to WT mice.

    Journal: Molecular cell

    Article Title: PKCα-LSD1-NF-κB-Signaling Cascade Is Crucial for Epigenetic Control of the Inflammatory Response.

    doi: 10.1016/j.molcel.2018.01.002

    Figure Lengend Snippet: Figure 7. Inhibition of PKCa Activity or LSD1 Activity in Mice Shows Attenuated Septic- Induced Mortality (A) Immunoblot analysis of lungs from WT mice (n = 3 per group). Mice were intraperitoneally injected with Go6976 (1 mg/kg body weight), GSK- LSD1 (1 mg/kg body weight), or an equal volume of vehicle for 1 hr prior to LPS challenge (10 mg/kg body weight) for 6 hr. (B) WT (blue line) or Lsd1SA/SA (red line) mice were subjected to CLP (n = 20 each). Animal survival was monitored every 6 hr for 144 hr after CLP. (C and D) WT mice (n = 20) were administered with Go6976 (green line) or an equal volume of vehicle (blue line) (C) or GSK-LSD1 (purple line) or an equal volume of vehicle (blue line) (D) after CLP (1 mg/kg body weight, each, intravenous [i.v.], at 12 and 50 hr after CLP). Animal survival was monitored every 6 hr for 144 hr after CLP. **p < 0.01 (log-rank test) (B–D). (E) Representative images of H&E staining of lung sections from WT and Lsd1SA/SA mice after CLP (n = 6 per group) or from WT mice intravenously in- jected with Go6976, GSK-LSD1, or vehicle (n = 6 per group, 1 mg/kg body weight, at 12 and 50 hr after CLP). Mice were euthanized 72 hr after CLP. Scale bars, 200 mm. (F and G) Plasma concentrations of cytokines (MCP-1, IL-6, and TNF-a) were measured 72 hr after CLP in WT and Lsd1SA/SA mice (F) or in WT mice intravenously injected (1 mg/kg body weight) with Go6976 or GSK-LSD1 at 12 and 50 hr after CLP (G). (H and I) Septic injury marker (ALT, LDH, and BUN) levels were measured 72 hr after CLP in WT and Lsd1SA/SA mice (H) or in WT mice intravenously in- jected (1 mg/kg body weight) with Go6976 or GSK- LSD1 at 12 and 50 hr after CLP (I). Data are expressed as mean ± SD; n = 5; *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA) (F and H). Data are expressed as mean ± SD; n = 5 per group; *p < 0.05, **p < 0.01 (unpaired two-tailed Student’s t test) (G and I). (J) Schematic model of the PKCa-LSD1-NF-kB- signaling axis showing that blocking PKCa or LSD1 activity decreases p65 protein stability and, thus, induces resistance to acute systemic inflammation, leading to increased survival rates of sepsis in Lsd1SA/SA mice compared to WT mice.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-p65 Santa Cruz sc-372 (ChIP-seq) ; RRID: AB_632037 Goat polyclonal anti-Lamin A/C Santa Cruz sc-6215 ; RRID: AB_648152 Rabbit polyclonal anti-GST Santa Cruz sc-459 ; RRID: AB_631586 Mouse monoclonal anti-GFP Santa Cruz sc-9996 ; RRID: AB_627695 Rabbit polyclonal anti-LSD1 Cell Signaling #2139 ; RRID: AB_2070135 Rabbit polyclonal anti-PKCa Cell Signaling #2056S ; RRID: AB_2284227 Rabbit polyclonal anti-p65 abcam ab7970 ; RRID: AB_306184 Rabbit polyclonal anti-LSD1 abcam ab17721 (ChIP-seq) ; RRID: AB_443964 Rabbit polyclonal anti-H3K9Ac abcam ab4441 ; RRID: AB_2118292 Rabbit polyclonal anti-H3K9me2 abcam ab1220 ; RRID: AB_449854 Rabbit polyclonal anti-H3K4me2 abcam ab32356 ; RRID: AB_732924 Rabbit polyclonal anti-LSD1 NOVUS NB-100-1762 ; RRID: AB_10001568 Rabbit polyclonal anti-PKCa NOVUS NB-110-57356 ; RRID: AB_844168 Rabbit polyclonal anti-C/EBPd NOVUS NB-110-85519 ; RRID: AB_1201191 Rabbit polyclonal anti-phospho(S657)-PKCa Millipore #06-822 ; RRID: AB_11213245 Rabbit polyclonal anti-phospho(S112)-LSD1 Millipore ABE 1462 ; RRID: N/A Mouse monoclonal anti-HA Covance MMS-101R ; RRID: AB_291263 Mouse monoclonal anti-b-actin Sigma A1978 ; RRID: AB_476692 Rabbit polyclonal anti-Tubulin AbFrontier LF-PA0146A ; RRID: N/A Rabbit polyclonal anti-methyl(K314/315)-p65 Elabscience biotechnology ENH006 ; RRID: N/A Mouse monoclonal anti-FK2 Enzo Life Sciences BML-PW8810 ; RRID: AB_10541840 Bacterial and Virus Strains DH5-a Escherichia coli Enzynomics CP010 Rossetta Escherichia coli Novagen 70954-3CN M15 pREP4 Escherichia coli QIAGEN 32149 Chemicals, Peptides, and Recombinant Proteins Lipopolysaccharides (LPS) from Escherichia coli Sigma L3129 GSK-LSD1 Sigma SML1072 Go6976 Cayman 13310 MG132 A.G. Scientific M-1157 DMSO (Vehicle) Sigma D2650 Ni-NTA Agarose QIAGEN 30210 cOmplete Protease Inhibitor Cocktail Roche 11697498001 TNT T7 Quick Coupled Transcription/Translation System Promega L1170 l-phosphatase NEB P0753 Human p65 protein (TNT) This paper N/A Human GST-LSD1 protein (GST purification) This paper N/A Human GST-p65 protein (GST purification) This paper N/A Human GST-SET7/9 protein (GST purification) This paper N/A Critical Commercial Assays TruSeq RNA Sample prep kit v2 Illumina RS-122-2001 Illumina Hi-seq 3000/4000 SBS kit v3 Illumina FC-410-1003 (Continued on next page) e1 Molecular Cell 69, 398–411.e1–e6, February 1, 2018

    Techniques: Inhibition, Activity Assay, Western Blot, Injection, Staining, Clinical Proteomics, Marker, Two Tailed Test, Blocking Assay

    WipB is targeted to lysosomes by its C-terminal domain where it interacts with components of the lysosomal nutrient sensing system ( a ) HeLa cells expressing GFP-fusion proteins of WipB, WipB D118A , WipB 1-364 and WipB 365-524 . Scale bar, 10 µm. ( b ) HeLa cells expressing GFP-WipB (green) stained using an anti-LAMP-1 antibody (Magenta) after permeabilisation and fixation. Scale bar, 10 µm. ( c ) HeLa cells expressing GFP-WipB (green) were incubated with Lysotracker (red) for 15 min before fixation. Scale bar, 10 µm. ( d ) SDS-PAGE of fractions following co-immunoprecipitation of GFP, GFP-WipB or the indicated GFP-WipB derivatives from HeLa cell lysates using anti-GFP antibody and immunoblotting with anti-v-ATPase A, -v-ATPase B or LAMTOR1 antibodies. A cropped blot is here displayed and the corresponding full-length blot is included in the supplementary information. ( e ) SDS-PAGE of fractions following co-immunoprecipitation of v-ATPase A, v-ATPase B or LAMTOR1 from lysates of HeLa cells expressing GFP or GFP-WipB and immunoblotting with anti-GFP antibody. A cropped blot is here displayed and the corresponding full-length blot is included in the supplementary information.

    Journal: Scientific Reports

    Article Title: The Legionella effector WipB is a translocated Ser/Thr phosphatase that targets the host lysosomal nutrient sensing machinery

    doi: 10.1038/s41598-017-10249-6

    Figure Lengend Snippet: WipB is targeted to lysosomes by its C-terminal domain where it interacts with components of the lysosomal nutrient sensing system ( a ) HeLa cells expressing GFP-fusion proteins of WipB, WipB D118A , WipB 1-364 and WipB 365-524 . Scale bar, 10 µm. ( b ) HeLa cells expressing GFP-WipB (green) stained using an anti-LAMP-1 antibody (Magenta) after permeabilisation and fixation. Scale bar, 10 µm. ( c ) HeLa cells expressing GFP-WipB (green) were incubated with Lysotracker (red) for 15 min before fixation. Scale bar, 10 µm. ( d ) SDS-PAGE of fractions following co-immunoprecipitation of GFP, GFP-WipB or the indicated GFP-WipB derivatives from HeLa cell lysates using anti-GFP antibody and immunoblotting with anti-v-ATPase A, -v-ATPase B or LAMTOR1 antibodies. A cropped blot is here displayed and the corresponding full-length blot is included in the supplementary information. ( e ) SDS-PAGE of fractions following co-immunoprecipitation of v-ATPase A, v-ATPase B or LAMTOR1 from lysates of HeLa cells expressing GFP or GFP-WipB and immunoblotting with anti-GFP antibody. A cropped blot is here displayed and the corresponding full-length blot is included in the supplementary information.

    Article Snippet: CHIP grade rabbit anti-GFP (Invitrogen) was used for co-immunoprecipitation and mouse anti-GFP (clontech) for Western-blotting.

    Techniques: Expressing, Staining, Incubation, SDS Page, Immunoprecipitation, Western Blot