immunoprecipitation buffer  (Thermo Fisher)


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    Name:
    Pierce IP Lysis Buffer
    Description:
    Thermo Scientific Pierce IP Lysis Buffer is optimized for cell lysate yield purity and compatibility with immunoprecipitation IP and Co IP as the downstream application for the cell lysate Features of IP Lysis Buffer • Optimized for compatibility with immunoprecipitation and pull down assays • Compatible with protein assays reporter assays and immunoassay procedures • Ready made formula is effective for extracting cytoplasmic membrane and nuclear proteins • Gentle formulation helps maintain protein complexes for co immunoprecipitation • Does not liberate genomic DNA which can cause high sample viscosity IP Lysis Buffer is a mammalian whole cell lysis reagent based on a modified RIPA buffer formulation without SDS This moderate strength lysis buffer effectively solubilizes cellular proteins but does not liberate genomic DNA or disrupt protein complexes like ordinary RIPA buffer Pierce IP Lysis Buffer is specially formulated for pull down and immunoprecipitation assays Pierce IP Lysis Buffer is effective for lysing cultured mammalian cells from both plated cells and cells pelleted from suspension cultures Optimized for pull down and immunoprecipitation assays this lysis buffer is also compatible with many other applications including the Thermo Scientific Pierce BCA and 660 nm Protein Assays protein purification and immunoassays e g ELISA Western blot Pierce IP Lysis Buffer is composed of 25 mM Tris HCl pH 7 4 150 mM NaCl 1 mM EDTA 1 NP 40 and 5 glycerol The buffer does not contain protease or phosphatase inhibitors however if desired inhibitors such as Thermo Scientific Halt Protease Inhibitor Cocktail or Phosphatase Inhibitor Cocktail can be added just before use to prevent proteolysis and maintain phosphorylation of proteins Related Products M PER Mammalian Protein Extraction Reagent RIPA Lysis and Extraction Buffer
    Catalog Number:
    87787
    Price:
    None
    Category:
    Lab Reagents and Chemicals
    Applications:
    Cell Lysis|Cell Lysis & Fractionation|Protein Biology|Protein Purification & Isolation
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    Structured Review

    Thermo Fisher immunoprecipitation buffer
    TRAPP-associated proteins were identified by <t>Immunoprecipitation</t> of TRS33 A. Experimental work flow for the S table I sotope L abeling I mmunoprecipitation M ass S pectrometry (SILIP-MS) in Arabidopsis. B. MS1 spectra show the enrichment of TRIPP in SILIP-MS of TRS33:TRS33-4MycHis/ trs33-1 versu Wild type (Col-0) control, and no enrichment of ClpC, a non-specific protein. Blue and red rows point to monoisotopic peaks of 14 N- and 15 N-labeled peptides, respectively.
    Thermo Scientific Pierce IP Lysis Buffer is optimized for cell lysate yield purity and compatibility with immunoprecipitation IP and Co IP as the downstream application for the cell lysate Features of IP Lysis Buffer • Optimized for compatibility with immunoprecipitation and pull down assays • Compatible with protein assays reporter assays and immunoassay procedures • Ready made formula is effective for extracting cytoplasmic membrane and nuclear proteins • Gentle formulation helps maintain protein complexes for co immunoprecipitation • Does not liberate genomic DNA which can cause high sample viscosity IP Lysis Buffer is a mammalian whole cell lysis reagent based on a modified RIPA buffer formulation without SDS This moderate strength lysis buffer effectively solubilizes cellular proteins but does not liberate genomic DNA or disrupt protein complexes like ordinary RIPA buffer Pierce IP Lysis Buffer is specially formulated for pull down and immunoprecipitation assays Pierce IP Lysis Buffer is effective for lysing cultured mammalian cells from both plated cells and cells pelleted from suspension cultures Optimized for pull down and immunoprecipitation assays this lysis buffer is also compatible with many other applications including the Thermo Scientific Pierce BCA and 660 nm Protein Assays protein purification and immunoassays e g ELISA Western blot Pierce IP Lysis Buffer is composed of 25 mM Tris HCl pH 7 4 150 mM NaCl 1 mM EDTA 1 NP 40 and 5 glycerol The buffer does not contain protease or phosphatase inhibitors however if desired inhibitors such as Thermo Scientific Halt Protease Inhibitor Cocktail or Phosphatase Inhibitor Cocktail can be added just before use to prevent proteolysis and maintain phosphorylation of proteins Related Products M PER Mammalian Protein Extraction Reagent RIPA Lysis and Extraction Buffer
    https://www.bioz.com/result/immunoprecipitation buffer/product/Thermo Fisher
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    Images

    1) Product Images from "Proteomic studies of the Arabidopsis TRAPP complexes reveal conserved organization and a novel plant-specific component with a role in plant development"

    Article Title: Proteomic studies of the Arabidopsis TRAPP complexes reveal conserved organization and a novel plant-specific component with a role in plant development

    Journal: bioRxiv

    doi: 10.1101/684258

    TRAPP-associated proteins were identified by Immunoprecipitation of TRS33 A. Experimental work flow for the S table I sotope L abeling I mmunoprecipitation M ass S pectrometry (SILIP-MS) in Arabidopsis. B. MS1 spectra show the enrichment of TRIPP in SILIP-MS of TRS33:TRS33-4MycHis/ trs33-1 versu Wild type (Col-0) control, and no enrichment of ClpC, a non-specific protein. Blue and red rows point to monoisotopic peaks of 14 N- and 15 N-labeled peptides, respectively.
    Figure Legend Snippet: TRAPP-associated proteins were identified by Immunoprecipitation of TRS33 A. Experimental work flow for the S table I sotope L abeling I mmunoprecipitation M ass S pectrometry (SILIP-MS) in Arabidopsis. B. MS1 spectra show the enrichment of TRIPP in SILIP-MS of TRS33:TRS33-4MycHis/ trs33-1 versu Wild type (Col-0) control, and no enrichment of ClpC, a non-specific protein. Blue and red rows point to monoisotopic peaks of 14 N- and 15 N-labeled peptides, respectively.

    Techniques Used: Immunoprecipitation, Labeling

    TRIPP interacts with TRAPPII subunits. A. Metabolic stable isotope labeling immunoprecipitation mass spectrometry (SILIP-MS) analysis of TRIPP identifies only TRAPPII subunits. SILIP-MS was performed using 35S:TRIPP-YFP / tripp-1 complemented plants. Plot shows log 2 ratio of signal intensities between samples labelled with light (L, 14 N) and heavy (H, 15 N) isotopes (L/H ratio) for two biological replicates, in which either the Col-0 control (Forward) or the TRIPP-YFP sample (Reciprocal) was labelled with 15 N. Note that TRAPP-III subunits are not identified in the data set. B. Representative spectra of an AtTRS120 peptide quantified in the SILIP-MS experiments of 14 N-TRIPP-YFP vs 15 N-Col-0 (upper panel) and 14 N-Col-0 vs 15 N-TRIP-YFP (lower panel). Blue and red arrow points to mono-isotopic peak of the 14 N and 15 N labeled peptide respectively. C. Representative spectra of an AtTRS130 peptide quantified in the TRIPP-YFP SILIP-MS experiments as descried for panel B. D. Yeast two-hybrid assays of interactions between TRIPP and TRAPP subunits. The panels are from different plates. Four independent replicate experiments were performed. The results show interactions of TRIPP with both T1 and T3 regions of AtTRS120 and with the plant specific C2/C3_DB AtTRS130 regions. T2_DB is not included as it is an auto activator, as evidenced by colony growth with the empt AD vector, and this precludes our ability to determine whether AtTRS120_T2 interacts with TRIPP. E. TRAPPII coding regions used for yeast two-hybrid interaction assays. Segments colored in red are conserved across kingdoms, while those in green are plant-specific. The orange moiety of the C2 segment is poorly conserved across kingdoms. The T2 middle segment corresponds to sequences found to interact with the exocyst in a yeast two-hybrid screen ( Rybak et al., 2014 ).
    Figure Legend Snippet: TRIPP interacts with TRAPPII subunits. A. Metabolic stable isotope labeling immunoprecipitation mass spectrometry (SILIP-MS) analysis of TRIPP identifies only TRAPPII subunits. SILIP-MS was performed using 35S:TRIPP-YFP / tripp-1 complemented plants. Plot shows log 2 ratio of signal intensities between samples labelled with light (L, 14 N) and heavy (H, 15 N) isotopes (L/H ratio) for two biological replicates, in which either the Col-0 control (Forward) or the TRIPP-YFP sample (Reciprocal) was labelled with 15 N. Note that TRAPP-III subunits are not identified in the data set. B. Representative spectra of an AtTRS120 peptide quantified in the SILIP-MS experiments of 14 N-TRIPP-YFP vs 15 N-Col-0 (upper panel) and 14 N-Col-0 vs 15 N-TRIP-YFP (lower panel). Blue and red arrow points to mono-isotopic peak of the 14 N and 15 N labeled peptide respectively. C. Representative spectra of an AtTRS130 peptide quantified in the TRIPP-YFP SILIP-MS experiments as descried for panel B. D. Yeast two-hybrid assays of interactions between TRIPP and TRAPP subunits. The panels are from different plates. Four independent replicate experiments were performed. The results show interactions of TRIPP with both T1 and T3 regions of AtTRS120 and with the plant specific C2/C3_DB AtTRS130 regions. T2_DB is not included as it is an auto activator, as evidenced by colony growth with the empt AD vector, and this precludes our ability to determine whether AtTRS120_T2 interacts with TRIPP. E. TRAPPII coding regions used for yeast two-hybrid interaction assays. Segments colored in red are conserved across kingdoms, while those in green are plant-specific. The orange moiety of the C2 segment is poorly conserved across kingdoms. The T2 middle segment corresponds to sequences found to interact with the exocyst in a yeast two-hybrid screen ( Rybak et al., 2014 ).

    Techniques Used: Labeling, Immunoprecipitation, Mass Spectrometry, Plasmid Preparation, Two Hybrid Screening

    2) Product Images from "Inhibiting DDX3X triggers tumor-intrinsic type I interferon response and enhances anti-tumor immunity"

    Article Title: Inhibiting DDX3X triggers tumor-intrinsic type I interferon response and enhances anti-tumor immunity

    Journal: bioRxiv

    doi: 10.1101/2020.09.09.289587

    DDX3X interacts with cytoplasmic ADAR1 and prevents the buildup of the cytoplasmic dsRNA. a , J2 dot blot analysis of RNA extracts from cytoplasmic or nucleus fractions of the DDX3X-control or -KD MCF7 cells. Electrophoresis analysis of RNAs isolated from cytoplasmic or nucleus fractions. A circle graph shows relative dot intensity (each cells’ cytoplasm + nucleus = 100%). b , Localization of DDX3X (upper) and levels of dsRNAs (lower) in cytoplasmic or nucleus fractions after leptomycin B (LMB; 30nM, 16 h) treatment. c , J2 Immunoprecipitation with whole MCF7 cell extracts with or without 5-AzaC treatment. MCF7 cells were fixed with 1 % formaldehyde and IP was performed. dsRNA-bound proteins were analyzed using western blot with anti-DDX3X or anti-IgG (light chain) antibodies. d , Interaction of DDX3X and ADAR. Immunoprecipitation of DDX3X from whole cell extracts or cytoplasmic fraction of MCF7 cells. IP was performed with DDX3X antibody or control IgG, respectively. Western blot analyzed using anti-ADAR1 and anti-DDX3X antibodies. e – g , mRNA expression of dsRNA sensing genes ( e ), ISGs ( f ), and IFNB1 ( g ) in DDX3X and ADAR1 single, or double KD cells. h , Determination of ADAR1 editing as the ratio between luminescence from Nluc/FFL expressed reporter plasmid. Data was calculated a relative response ratio (RRR) = (experimental sample ratio-negative control ratio) / (positive control ratio-negative control ratio)). Data are representative of three independent experiments. Data represented as mean ± SEM. Statistics were calculated using unpaired t-tests. * P
    Figure Legend Snippet: DDX3X interacts with cytoplasmic ADAR1 and prevents the buildup of the cytoplasmic dsRNA. a , J2 dot blot analysis of RNA extracts from cytoplasmic or nucleus fractions of the DDX3X-control or -KD MCF7 cells. Electrophoresis analysis of RNAs isolated from cytoplasmic or nucleus fractions. A circle graph shows relative dot intensity (each cells’ cytoplasm + nucleus = 100%). b , Localization of DDX3X (upper) and levels of dsRNAs (lower) in cytoplasmic or nucleus fractions after leptomycin B (LMB; 30nM, 16 h) treatment. c , J2 Immunoprecipitation with whole MCF7 cell extracts with or without 5-AzaC treatment. MCF7 cells were fixed with 1 % formaldehyde and IP was performed. dsRNA-bound proteins were analyzed using western blot with anti-DDX3X or anti-IgG (light chain) antibodies. d , Interaction of DDX3X and ADAR. Immunoprecipitation of DDX3X from whole cell extracts or cytoplasmic fraction of MCF7 cells. IP was performed with DDX3X antibody or control IgG, respectively. Western blot analyzed using anti-ADAR1 and anti-DDX3X antibodies. e – g , mRNA expression of dsRNA sensing genes ( e ), ISGs ( f ), and IFNB1 ( g ) in DDX3X and ADAR1 single, or double KD cells. h , Determination of ADAR1 editing as the ratio between luminescence from Nluc/FFL expressed reporter plasmid. Data was calculated a relative response ratio (RRR) = (experimental sample ratio-negative control ratio) / (positive control ratio-negative control ratio)). Data are representative of three independent experiments. Data represented as mean ± SEM. Statistics were calculated using unpaired t-tests. * P

    Techniques Used: Dot Blot, Electrophoresis, Isolation, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Negative Control, Positive Control

    3) Product Images from "The ubiquitin ligase HECTD1 promotes retinoic acid signaling required for development of the aortic arch"

    Article Title: The ubiquitin ligase HECTD1 promotes retinoic acid signaling required for development of the aortic arch

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.036491

    HECTD1 binds to RARA and influences its ubiquitination. (A) HA-RARA(281-462), binds to Myc-HECTD1(1-551) expressed in rabbit reticulocyte lysates. Proteins were immunoprecipitated (IP) as indicated followed by immunoblotting (IB) with streptavidin (STV). (B) RARA binds to HA-HECTD1 and ligase-deficient HA-HECTD1 C2579G (HA-HECTD11 CG ), but not to HA-NEDD4, another HECT domain-containing E3 ligase, in HEK293T cells. WCL, whole-cell lysate. (C) RARA binds HECTD1 in vivo in embryo lysates prepared from E10.5 wild-type and Hectd1 XC/XC mutant embryos. The epitope recognized by the HECTD1 antibody used for immunoprecipitation is not present in Hectd1 opm/opm mutant embryos, thus the failure of HECTD1 opm to pull down RARA serves as a negative control. (D) HECTD1 influences ubiquitination of RARA. HEK293T cells were transfected with Myc-RARA and either HA-HECTD1 or ligase-deficient HECTD1 C2579G . RARA was immunoprecipitated followed by immunoblotting with the FK2 antibody to detect poly- and monoubiquitinated RARA, or FK1 antibody to detect polyubiquitinated RARA. FK2 immunostaining indicates a slight increase in ubiquitinated RARA levels with expression of either wild-type or ligase-defective HECTD1, whereas FK1 immunostaining shows reduced ubiquitinated RARA in HA-HECTD1 C2579G transfected cells, consistent with the predicted dominant-negative activity of this construct. (E) Knockdown of HECTD1 by siRNA in HEK293T cells results in decreased ubiquitinated RARA. (F) MEFs derived from Hectd1 opm/opm and Hectd1 XC/XC mutant embryos demonstrate reduced ubiquitinated proteins in RARA immunoprecipitates and increased RARA levels in mutant compared with wild-type cells. Key positive signals are highlighted in boxes.
    Figure Legend Snippet: HECTD1 binds to RARA and influences its ubiquitination. (A) HA-RARA(281-462), binds to Myc-HECTD1(1-551) expressed in rabbit reticulocyte lysates. Proteins were immunoprecipitated (IP) as indicated followed by immunoblotting (IB) with streptavidin (STV). (B) RARA binds to HA-HECTD1 and ligase-deficient HA-HECTD1 C2579G (HA-HECTD11 CG ), but not to HA-NEDD4, another HECT domain-containing E3 ligase, in HEK293T cells. WCL, whole-cell lysate. (C) RARA binds HECTD1 in vivo in embryo lysates prepared from E10.5 wild-type and Hectd1 XC/XC mutant embryos. The epitope recognized by the HECTD1 antibody used for immunoprecipitation is not present in Hectd1 opm/opm mutant embryos, thus the failure of HECTD1 opm to pull down RARA serves as a negative control. (D) HECTD1 influences ubiquitination of RARA. HEK293T cells were transfected with Myc-RARA and either HA-HECTD1 or ligase-deficient HECTD1 C2579G . RARA was immunoprecipitated followed by immunoblotting with the FK2 antibody to detect poly- and monoubiquitinated RARA, or FK1 antibody to detect polyubiquitinated RARA. FK2 immunostaining indicates a slight increase in ubiquitinated RARA levels with expression of either wild-type or ligase-defective HECTD1, whereas FK1 immunostaining shows reduced ubiquitinated RARA in HA-HECTD1 C2579G transfected cells, consistent with the predicted dominant-negative activity of this construct. (E) Knockdown of HECTD1 by siRNA in HEK293T cells results in decreased ubiquitinated RARA. (F) MEFs derived from Hectd1 opm/opm and Hectd1 XC/XC mutant embryos demonstrate reduced ubiquitinated proteins in RARA immunoprecipitates and increased RARA levels in mutant compared with wild-type cells. Key positive signals are highlighted in boxes.

    Techniques Used: Immunoprecipitation, In Vivo, Mutagenesis, Negative Control, Transfection, Immunostaining, Expressing, Dominant Negative Mutation, Activity Assay, Construct, Derivative Assay

    4) Product Images from "ARRB1 ameliorates liver ischaemia/reperfusion injury via antagonizing TRAF6‐mediated Lysine 6‐linked polyubiquitination of ASK1 in hepatocytes, et al. ARRB1 ameliorates liver ischaemia/reperfusion injury via antagonizing TRAF6‐mediated Lysine 6‐linked polyubiquitination of ASK1 in hepatocytes"

    Article Title: ARRB1 ameliorates liver ischaemia/reperfusion injury via antagonizing TRAF6‐mediated Lysine 6‐linked polyubiquitination of ASK1 in hepatocytes, et al. ARRB1 ameliorates liver ischaemia/reperfusion injury via antagonizing TRAF6‐mediated Lysine 6‐linked polyubiquitination of ASK1 in hepatocytes

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.15412

    ARRB1 interacts with ASK1 and then antagonizes its TRAF6‐mediated K6‐linked polyubiquitination, thus inhibiting its activation in hepatocytes during hepatic I/R injury. A, Immunoblotting detection of phosphorylated ASK1 protein (left panel) and co‐IP assay for the interaction between His‐ASK1 and HA‐TRAF proteins (right panel) in L02 hepatocytes stimulated with H/R for 6 h. B, His‐tagged ASK1 and HA‐tagged TRAF6 plasmids were cotransfected into L02 hepatocytes. Anti‐His antibody (left panel) and anti‐HA antibody (right panel) were used for immunoprecipitation. C, Immunoprecipitation analysis of indicated ubiquitination types of ASK1 in L02 hepatocytes transfected with HA‐TRAF6 or empty vector in respond to H/R insult or not. D, IP analysis was conducted to detect the binding association between ASK1 and ARRB1 in L02 hepatocytes under normal condition. His‐tagged ASK1 and Flag‐tagged ARRB1 plasmids were cotransfected into L02 hepatocytes. Anti‐His antibody (left panel) and anti‐Flag antibody (right panel) were used for immunoprecipitation. E, IP analysis was conducted to detect the binding association between ASK1 and ARRB1 in L02 hepatocytes when treated with H/R challenge. Anti‐His antibody (left panel) and anti‐Flag antibody (right panel) were used for immunoprecipitation. F, IP analysis showing the expression level of ARRB1 and binding capability between ASK1 and TRAF6 in L02 hepatocytes under H/R challenge or not. Anti‐His antibody (left panel) and anti‐HA antibody (right panel) were used for immunoprecipitation. G and H, Lysates of liver lobes challenged with or without I/R surgery (G) or whole‐cell lysates of primary hepatocytes subjected to or not H/R insult (H) were subjected to immunoprecipitation with anti‐ASK1 antibody followed by immunoblotting with anti‐K6‐linked polyubiquitination antibody when ARRB1 was overexpressed or knockout
    Figure Legend Snippet: ARRB1 interacts with ASK1 and then antagonizes its TRAF6‐mediated K6‐linked polyubiquitination, thus inhibiting its activation in hepatocytes during hepatic I/R injury. A, Immunoblotting detection of phosphorylated ASK1 protein (left panel) and co‐IP assay for the interaction between His‐ASK1 and HA‐TRAF proteins (right panel) in L02 hepatocytes stimulated with H/R for 6 h. B, His‐tagged ASK1 and HA‐tagged TRAF6 plasmids were cotransfected into L02 hepatocytes. Anti‐His antibody (left panel) and anti‐HA antibody (right panel) were used for immunoprecipitation. C, Immunoprecipitation analysis of indicated ubiquitination types of ASK1 in L02 hepatocytes transfected with HA‐TRAF6 or empty vector in respond to H/R insult or not. D, IP analysis was conducted to detect the binding association between ASK1 and ARRB1 in L02 hepatocytes under normal condition. His‐tagged ASK1 and Flag‐tagged ARRB1 plasmids were cotransfected into L02 hepatocytes. Anti‐His antibody (left panel) and anti‐Flag antibody (right panel) were used for immunoprecipitation. E, IP analysis was conducted to detect the binding association between ASK1 and ARRB1 in L02 hepatocytes when treated with H/R challenge. Anti‐His antibody (left panel) and anti‐Flag antibody (right panel) were used for immunoprecipitation. F, IP analysis showing the expression level of ARRB1 and binding capability between ASK1 and TRAF6 in L02 hepatocytes under H/R challenge or not. Anti‐His antibody (left panel) and anti‐HA antibody (right panel) were used for immunoprecipitation. G and H, Lysates of liver lobes challenged with or without I/R surgery (G) or whole‐cell lysates of primary hepatocytes subjected to or not H/R insult (H) were subjected to immunoprecipitation with anti‐ASK1 antibody followed by immunoblotting with anti‐K6‐linked polyubiquitination antibody when ARRB1 was overexpressed or knockout

    Techniques Used: Activation Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Binding Assay, Expressing, Knock-Out

    5) Product Images from "Expression of Multidrug Resistance Associated Protein 5 (MRP5) on Cornea and Its Role in Drug Efflux"

    Article Title: Expression of Multidrug Resistance Associated Protein 5 (MRP5) on Cornea and Its Role in Drug Efflux

    Journal: Journal of Ocular Pharmacology and Therapeutics

    doi: 10.1089/jop.2008.0084

    Immunoprecipitation followed by Western blot indicating the presence of MRP5 in membrane fraction of SV40-HCEC and MDCKII-MRP5 (+ve control).
    Figure Legend Snippet: Immunoprecipitation followed by Western blot indicating the presence of MRP5 in membrane fraction of SV40-HCEC and MDCKII-MRP5 (+ve control).

    Techniques Used: Immunoprecipitation, Western Blot

    6) Product Images from "NRF2 regulates serine biosynthesis in non-small cell lung cancer"

    Article Title: NRF2 regulates serine biosynthesis in non-small cell lung cancer

    Journal: Nature genetics

    doi: 10.1038/ng.3421

    NRF2 regulates the expression of serine/glycine biosynthesis genes through ATF4. (a) ATF4 mRNA expression in A549 cells expressing scramble shRNA (SCR), or NRF2 shRNA #1. (b) Western blot of NRF2, ATF4 and ACTIN expression in cells from (a). (c) Western blot of NRF2, ATF4 and serine pathway enzyme expression in lysates from A549s expressing scramble (SCR), NRF2 shRNA #1, or ATF4 shRNAs #1 or #2. (d) mRNA expression in cells from (c). (e) ATF4 knockdown impairs serine biosynthesis. Cell lines from were grown in the presence of U- 13 C-glucose for the indicated time points, the metabolites extracted and the fractional 13 C-labeling on serine analysed by LC/MS. (f) ATF4 rescues serine biosynthesis enzyme expression following NRF2 knockdown. A549 cells were infected with lentivirus encoding mATF4 prior to infection with scramble or NRF2-targeting lentivirus. (g) Western analysis of NRF2, ATF4, and ACTIN expression in the cells from (f). (h) ATF4 rescues the serine biosynthesis defect in shNRF2 A549 cells. Cells were assayed as in (e). (i) ATF4 rescues the growth of H1975 cells in serine deficient media. Cells expressing luciferase (LUC) or ATF4 were grown in the indicated media for 3 days and cell number normalized to cells grown in full media. (j) Chromatin immunoprecipitation of ATF4 to the PHGDH, PSAT1 and SHMT2 promoters. Samples were normalized to IgG control immunoprecipitations. Results are the average of 3 technical (a, d, f, j) or biological (e, h, i) replicates.
    Figure Legend Snippet: NRF2 regulates the expression of serine/glycine biosynthesis genes through ATF4. (a) ATF4 mRNA expression in A549 cells expressing scramble shRNA (SCR), or NRF2 shRNA #1. (b) Western blot of NRF2, ATF4 and ACTIN expression in cells from (a). (c) Western blot of NRF2, ATF4 and serine pathway enzyme expression in lysates from A549s expressing scramble (SCR), NRF2 shRNA #1, or ATF4 shRNAs #1 or #2. (d) mRNA expression in cells from (c). (e) ATF4 knockdown impairs serine biosynthesis. Cell lines from were grown in the presence of U- 13 C-glucose for the indicated time points, the metabolites extracted and the fractional 13 C-labeling on serine analysed by LC/MS. (f) ATF4 rescues serine biosynthesis enzyme expression following NRF2 knockdown. A549 cells were infected with lentivirus encoding mATF4 prior to infection with scramble or NRF2-targeting lentivirus. (g) Western analysis of NRF2, ATF4, and ACTIN expression in the cells from (f). (h) ATF4 rescues the serine biosynthesis defect in shNRF2 A549 cells. Cells were assayed as in (e). (i) ATF4 rescues the growth of H1975 cells in serine deficient media. Cells expressing luciferase (LUC) or ATF4 were grown in the indicated media for 3 days and cell number normalized to cells grown in full media. (j) Chromatin immunoprecipitation of ATF4 to the PHGDH, PSAT1 and SHMT2 promoters. Samples were normalized to IgG control immunoprecipitations. Results are the average of 3 technical (a, d, f, j) or biological (e, h, i) replicates.

    Techniques Used: Expressing, shRNA, Western Blot, Labeling, Liquid Chromatography with Mass Spectroscopy, Infection, Luciferase, Chromatin Immunoprecipitation

    7) Product Images from "Biological Effects and Use of PrPSc- and PrP-Specific Antibodies Generated by Immunization with Purified Full-Length Native Mouse Prions ▿"

    Article Title: Biological Effects and Use of PrPSc- and PrP-Specific Antibodies Generated by Immunization with Purified Full-Length Native Mouse Prions ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02467-10

    Immunoprecipitation of PrP Sc from CJD samples with MAb W261. (A) Detection of PrP Sc in various sporadic (s) cases and one genetic (g) case of CJD. (B) Detection of PrP Sc in various CJD cases, including vCJD. (C) Demonstration of various amounts of PrP
    Figure Legend Snippet: Immunoprecipitation of PrP Sc from CJD samples with MAb W261. (A) Detection of PrP Sc in various sporadic (s) cases and one genetic (g) case of CJD. (B) Detection of PrP Sc in various CJD cases, including vCJD. (C) Demonstration of various amounts of PrP

    Techniques Used: Immunoprecipitation

    Reaction patterns of PrP Sc -specific MAbs in immunoprecipitation (IP). (A) MAbs W68 and W261 react exclusively with the PrP Sc isoform. (B and C) Treatment of precipitates with PK reveals the presence of PK-resistant PrP Sc in MAb-precipitated material for
    Figure Legend Snippet: Reaction patterns of PrP Sc -specific MAbs in immunoprecipitation (IP). (A) MAbs W68 and W261 react exclusively with the PrP Sc isoform. (B and C) Treatment of precipitates with PK reveals the presence of PK-resistant PrP Sc in MAb-precipitated material for

    Techniques Used: Immunoprecipitation

    8) Product Images from "Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies"

    Article Title: Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026499

    Antibody specificity. A ) Sequence alignment of MeCP2 from different species. Identical residues are shaded in gray. The identities range from 93% (human-mouse) to 97% (rat-mouse). B ) For a multi-species immunoblot nuclear extracts from pig, mouse and rat brain (10 6 nuclei) were loaded and probed with the antibodies as indicated. C ) For immunoprecipitation analysis, mouse brain whole cell (for rat antibodies) and nuclei (for mouse antibodies) extracts were incubated with the monoclonal antibodies as indicated followed by western blot analysis.
    Figure Legend Snippet: Antibody specificity. A ) Sequence alignment of MeCP2 from different species. Identical residues are shaded in gray. The identities range from 93% (human-mouse) to 97% (rat-mouse). B ) For a multi-species immunoblot nuclear extracts from pig, mouse and rat brain (10 6 nuclei) were loaded and probed with the antibodies as indicated. C ) For immunoprecipitation analysis, mouse brain whole cell (for rat antibodies) and nuclei (for mouse antibodies) extracts were incubated with the monoclonal antibodies as indicated followed by western blot analysis.

    Techniques Used: Sequencing, Immunoprecipitation, Incubation, Western Blot

    Chromatin immunoprecipitation. Chromatin immunoprecipitation assays were performed using mouse brain nuclear extracts obtained from wild type mice and MeCP2 knock out (KO) mice as negative control. The anti histone H3 antibody was used as a positive control of chromatin immunoprecipitation assay efficiency. IgG was used as a negative control of chromatin immunoprecipitation.
    Figure Legend Snippet: Chromatin immunoprecipitation. Chromatin immunoprecipitation assays were performed using mouse brain nuclear extracts obtained from wild type mice and MeCP2 knock out (KO) mice as negative control. The anti histone H3 antibody was used as a positive control of chromatin immunoprecipitation assay efficiency. IgG was used as a negative control of chromatin immunoprecipitation.

    Techniques Used: Chromatin Immunoprecipitation, Mouse Assay, Knock-Out, Negative Control, Positive Control

    9) Product Images from "Structural protein 4.1R is integrally involved in nuclear envelope protein localization, centrosome-nucleus association and transcriptional signaling"

    Article Title: Structural protein 4.1R is integrally involved in nuclear envelope protein localization, centrosome-nucleus association and transcriptional signaling

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.077883

    4.1R and emerin co-immunoprecipitate and partially colocalize in human and murine fibroblasts. ( A ) Immunoprecipitation (IP) from HeLa whole cell sonicates. Clarified whole cell sonicates were prepared, immunoprecipitated with anti-4.1R antibody, and eluted
    Figure Legend Snippet: 4.1R and emerin co-immunoprecipitate and partially colocalize in human and murine fibroblasts. ( A ) Immunoprecipitation (IP) from HeLa whole cell sonicates. Clarified whole cell sonicates were prepared, immunoprecipitated with anti-4.1R antibody, and eluted

    Techniques Used: Immunoprecipitation

    10) Product Images from "Control of somatic tissue differentiation by the long non-coding RNA TINCR"

    Article Title: Control of somatic tissue differentiation by the long non-coding RNA TINCR

    Journal: Nature

    doi: 10.1038/nature11661

    TINCR interacts with differentiation mRNAs and STAU1 protein a , Enriched GO terms in TINCR-interacting genes detected by RIA-Seq. b , Protein microarray analysis detects TINCR RNA binding to STAU1 protein. Human recombinant protein microarray spotted with approximately 9,400 proteins (left); enlarged 144 protein spot subarray (middle) demonstrating strand-specific binding of TINCR sense strand to STAU1 protein (right); DUPD1 protein negative control is shown. Alexa-Fluor-647-labelled rabbit anti-mouse IgG in the top left corner of each subarray. c , STAU1 protein immunoprecipitation pulls down TINCR RNA. ANCR and LINC1 (also known as XIST ) represent lncRNA controls. d , Streptavidin precipitation of in vitro synthesized biotinylated TINCR RNA pulls down STAU1 protein. HA, haemagglutinin; WB, western blot.
    Figure Legend Snippet: TINCR interacts with differentiation mRNAs and STAU1 protein a , Enriched GO terms in TINCR-interacting genes detected by RIA-Seq. b , Protein microarray analysis detects TINCR RNA binding to STAU1 protein. Human recombinant protein microarray spotted with approximately 9,400 proteins (left); enlarged 144 protein spot subarray (middle) demonstrating strand-specific binding of TINCR sense strand to STAU1 protein (right); DUPD1 protein negative control is shown. Alexa-Fluor-647-labelled rabbit anti-mouse IgG in the top left corner of each subarray. c , STAU1 protein immunoprecipitation pulls down TINCR RNA. ANCR and LINC1 (also known as XIST ) represent lncRNA controls. d , Streptavidin precipitation of in vitro synthesized biotinylated TINCR RNA pulls down STAU1 protein. HA, haemagglutinin; WB, western blot.

    Techniques Used: Microarray, RNA Binding Assay, Recombinant, Binding Assay, Negative Control, Immunoprecipitation, In Vitro, Synthesized, Western Blot

    11) Product Images from "Intracellular Shuttling and Mitochondrial Function of Thioredoxin-interacting Protein *"

    Article Title: Intracellular Shuttling and Mitochondrial Function of Thioredoxin-interacting Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.034421

    TXNIP interaction with mitochondrial Trx2. A , effects of oxidative stress on TXNIP-Trx2 co-immunoprecipitation. INS-1 cells were treated without (control ( C )) or with H 2 O 2 (15 μ m for 4 h) prior to isolation of their mitochondrial fractions and
    Figure Legend Snippet: TXNIP interaction with mitochondrial Trx2. A , effects of oxidative stress on TXNIP-Trx2 co-immunoprecipitation. INS-1 cells were treated without (control ( C )) or with H 2 O 2 (15 μ m for 4 h) prior to isolation of their mitochondrial fractions and

    Techniques Used: Immunoprecipitation, Isolation

    12) Product Images from "The ubiquitin ligase HECTD1 promotes retinoic acid signaling required for development of the aortic arch"

    Article Title: The ubiquitin ligase HECTD1 promotes retinoic acid signaling required for development of the aortic arch

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.036491

    HECTD1 binds to RARA and influences its ubiquitination. (A) HA-RARA(281-462), binds to Myc-HECTD1(1-551) expressed in rabbit reticulocyte lysates. Proteins were immunoprecipitated (IP) as indicated followed by immunoblotting (IB) with streptavidin (STV). (B) RARA binds to HA-HECTD1 and ligase-deficient HA-HECTD1 C2579G (HA-HECTD11 CG ), but not to HA-NEDD4, another HECT domain-containing E3 ligase, in HEK293T cells. WCL, whole-cell lysate. (C) RARA binds HECTD1 in vivo in embryo lysates prepared from E10.5 wild-type and Hectd1 XC/XC mutant embryos. The epitope recognized by the HECTD1 antibody used for immunoprecipitation is not present in Hectd1 opm/opm mutant embryos, thus the failure of HECTD1 opm to pull down RARA serves as a negative control. (D) HECTD1 influences ubiquitination of RARA. HEK293T cells were transfected with Myc-RARA and either HA-HECTD1 or ligase-deficient HECTD1 C2579G . RARA was immunoprecipitated followed by immunoblotting with the FK2 antibody to detect poly- and monoubiquitinated RARA, or FK1 antibody to detect polyubiquitinated RARA. FK2 immunostaining indicates a slight increase in ubiquitinated RARA levels with expression of either wild-type or ligase-defective HECTD1, whereas FK1 immunostaining shows reduced ubiquitinated RARA in HA-HECTD1 C2579G transfected cells, consistent with the predicted dominant-negative activity of this construct. (E) Knockdown of HECTD1 by siRNA in HEK293T cells results in decreased ubiquitinated RARA. (F) MEFs derived from Hectd1 opm/opm and Hectd1 XC/XC mutant embryos demonstrate reduced ubiquitinated proteins in RARA immunoprecipitates and increased RARA levels in mutant compared with wild-type cells. Key positive signals are highlighted in boxes.
    Figure Legend Snippet: HECTD1 binds to RARA and influences its ubiquitination. (A) HA-RARA(281-462), binds to Myc-HECTD1(1-551) expressed in rabbit reticulocyte lysates. Proteins were immunoprecipitated (IP) as indicated followed by immunoblotting (IB) with streptavidin (STV). (B) RARA binds to HA-HECTD1 and ligase-deficient HA-HECTD1 C2579G (HA-HECTD11 CG ), but not to HA-NEDD4, another HECT domain-containing E3 ligase, in HEK293T cells. WCL, whole-cell lysate. (C) RARA binds HECTD1 in vivo in embryo lysates prepared from E10.5 wild-type and Hectd1 XC/XC mutant embryos. The epitope recognized by the HECTD1 antibody used for immunoprecipitation is not present in Hectd1 opm/opm mutant embryos, thus the failure of HECTD1 opm to pull down RARA serves as a negative control. (D) HECTD1 influences ubiquitination of RARA. HEK293T cells were transfected with Myc-RARA and either HA-HECTD1 or ligase-deficient HECTD1 C2579G . RARA was immunoprecipitated followed by immunoblotting with the FK2 antibody to detect poly- and monoubiquitinated RARA, or FK1 antibody to detect polyubiquitinated RARA. FK2 immunostaining indicates a slight increase in ubiquitinated RARA levels with expression of either wild-type or ligase-defective HECTD1, whereas FK1 immunostaining shows reduced ubiquitinated RARA in HA-HECTD1 C2579G transfected cells, consistent with the predicted dominant-negative activity of this construct. (E) Knockdown of HECTD1 by siRNA in HEK293T cells results in decreased ubiquitinated RARA. (F) MEFs derived from Hectd1 opm/opm and Hectd1 XC/XC mutant embryos demonstrate reduced ubiquitinated proteins in RARA immunoprecipitates and increased RARA levels in mutant compared with wild-type cells. Key positive signals are highlighted in boxes.

    Techniques Used: Immunoprecipitation, In Vivo, Mutagenesis, Negative Control, Transfection, Immunostaining, Expressing, Dominant Negative Mutation, Activity Assay, Construct, Derivative Assay

    13) Product Images from "Inhibiting DDX3X triggers tumor-intrinsic type I interferon response and enhances anti-tumor immunity"

    Article Title: Inhibiting DDX3X triggers tumor-intrinsic type I interferon response and enhances anti-tumor immunity

    Journal: bioRxiv

    doi: 10.1101/2020.09.09.289587

    DDX3X interacts with cytoplasmic ADAR1 and prevents the buildup of the cytoplasmic dsRNA. a , J2 dot blot analysis of RNA extracts from cytoplasmic or nucleus fractions of the DDX3X-control or -KD MCF7 cells. Electrophoresis analysis of RNAs isolated from cytoplasmic or nucleus fractions. A circle graph shows relative dot intensity (each cells’ cytoplasm + nucleus = 100%). b , Localization of DDX3X (upper) and levels of dsRNAs (lower) in cytoplasmic or nucleus fractions after leptomycin B (LMB; 30nM, 16 h) treatment. c , J2 Immunoprecipitation with whole MCF7 cell extracts with or without 5-AzaC treatment. MCF7 cells were fixed with 1 % formaldehyde and IP was performed. dsRNA-bound proteins were analyzed using western blot with anti-DDX3X or anti-IgG (light chain) antibodies. d , Interaction of DDX3X and ADAR. Immunoprecipitation of DDX3X from whole cell extracts or cytoplasmic fraction of MCF7 cells. IP was performed with DDX3X antibody or control IgG, respectively. Western blot analyzed using anti-ADAR1 and anti-DDX3X antibodies. e – g , mRNA expression of dsRNA sensing genes ( e ), ISGs ( f ), and IFNB1 ( g ) in DDX3X and ADAR1 single, or double KD cells. h , Determination of ADAR1 editing as the ratio between luminescence from Nluc/FFL expressed reporter plasmid. Data was calculated a relative response ratio (RRR) = (experimental sample ratio-negative control ratio) / (positive control ratio-negative control ratio)). Data are representative of three independent experiments. Data represented as mean ± SEM. Statistics were calculated using unpaired t-tests. * P
    Figure Legend Snippet: DDX3X interacts with cytoplasmic ADAR1 and prevents the buildup of the cytoplasmic dsRNA. a , J2 dot blot analysis of RNA extracts from cytoplasmic or nucleus fractions of the DDX3X-control or -KD MCF7 cells. Electrophoresis analysis of RNAs isolated from cytoplasmic or nucleus fractions. A circle graph shows relative dot intensity (each cells’ cytoplasm + nucleus = 100%). b , Localization of DDX3X (upper) and levels of dsRNAs (lower) in cytoplasmic or nucleus fractions after leptomycin B (LMB; 30nM, 16 h) treatment. c , J2 Immunoprecipitation with whole MCF7 cell extracts with or without 5-AzaC treatment. MCF7 cells were fixed with 1 % formaldehyde and IP was performed. dsRNA-bound proteins were analyzed using western blot with anti-DDX3X or anti-IgG (light chain) antibodies. d , Interaction of DDX3X and ADAR. Immunoprecipitation of DDX3X from whole cell extracts or cytoplasmic fraction of MCF7 cells. IP was performed with DDX3X antibody or control IgG, respectively. Western blot analyzed using anti-ADAR1 and anti-DDX3X antibodies. e – g , mRNA expression of dsRNA sensing genes ( e ), ISGs ( f ), and IFNB1 ( g ) in DDX3X and ADAR1 single, or double KD cells. h , Determination of ADAR1 editing as the ratio between luminescence from Nluc/FFL expressed reporter plasmid. Data was calculated a relative response ratio (RRR) = (experimental sample ratio-negative control ratio) / (positive control ratio-negative control ratio)). Data are representative of three independent experiments. Data represented as mean ± SEM. Statistics were calculated using unpaired t-tests. * P

    Techniques Used: Dot Blot, Electrophoresis, Isolation, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Negative Control, Positive Control

    14) Product Images from "Hydroxyurea-inducible SAR1 gene acts through the Giα/JNK/Jun pathway to regulate γ-globin expression"

    Article Title: Hydroxyurea-inducible SAR1 gene acts through the Giα/JNK/Jun pathway to regulate γ-globin expression

    Journal: Blood

    doi: 10.1182/blood-2013-10-534842

    HU activates NF-κB signaling and enhances NF-κB binding to the SAR1 promoter region. (A) EMSA for the Elk-1/NF-κB was performed using K562 nuclear extracts (10 μg) and oligonucleotide probes containing either a wild-type or mutant Elk-1/NF-κB–binding site. Competition analysis was performed in the presence of 10-, 100-, or 500-fold excess of unlabeled oligonucleotides (right panel). Antibody-supershift assays were performed using antibodies against NF-κB p50, c-Rel, and Elk-1. Two Elk-1/NF-κB–specific DNA-protein complexes are indicated as A and B. The DNA-protein complex supershifted by anti-NF-κB p50 antibody is indicated as ss. (B) EMSA analysis of the effects of HU on NF-κB binding to its recognition site in the SAR1 promoter. EMSA was performed using nuclear extracts (10 μg) isolated from K562 cells treated with 100μM HU for the indicated period and oligonucleotide probes containing the Elk-1/NF-κB–binding site. A and B represent Elk-1/NF-κB–specific DNA-protein complexes as described in A. (C) At day 6 of differentiation, CD34 + cells were treated with or without 100 µM HU. Cells were then harvested and subjected to ChIP assay using antibody against NF-κB or Elk-1 to immunoprecipitate chromatin-protein complexes. A parallel ChIP assay was performed using rabbit IgG for the immunoprecipitation step as a ChIP assay negative control. DNA was amplified and quantitated by PCR with specific primers flanking the SAR1 gene promoter from −137 to −12. (D) CD34 + cells were treated in the presence or absence of 100 μM HU from day 4 to day 7 of differentiation, and preincubated in medium with 0, 5, 7.5, or 10 μg/mL BAY11-7082 for 30 minutes at day 6, transfected with a construct containing the −977 to +49 region of the SAR1 gene, then assayed for luciferase activity 24 hours after transfection. * P
    Figure Legend Snippet: HU activates NF-κB signaling and enhances NF-κB binding to the SAR1 promoter region. (A) EMSA for the Elk-1/NF-κB was performed using K562 nuclear extracts (10 μg) and oligonucleotide probes containing either a wild-type or mutant Elk-1/NF-κB–binding site. Competition analysis was performed in the presence of 10-, 100-, or 500-fold excess of unlabeled oligonucleotides (right panel). Antibody-supershift assays were performed using antibodies against NF-κB p50, c-Rel, and Elk-1. Two Elk-1/NF-κB–specific DNA-protein complexes are indicated as A and B. The DNA-protein complex supershifted by anti-NF-κB p50 antibody is indicated as ss. (B) EMSA analysis of the effects of HU on NF-κB binding to its recognition site in the SAR1 promoter. EMSA was performed using nuclear extracts (10 μg) isolated from K562 cells treated with 100μM HU for the indicated period and oligonucleotide probes containing the Elk-1/NF-κB–binding site. A and B represent Elk-1/NF-κB–specific DNA-protein complexes as described in A. (C) At day 6 of differentiation, CD34 + cells were treated with or without 100 µM HU. Cells were then harvested and subjected to ChIP assay using antibody against NF-κB or Elk-1 to immunoprecipitate chromatin-protein complexes. A parallel ChIP assay was performed using rabbit IgG for the immunoprecipitation step as a ChIP assay negative control. DNA was amplified and quantitated by PCR with specific primers flanking the SAR1 gene promoter from −137 to −12. (D) CD34 + cells were treated in the presence or absence of 100 μM HU from day 4 to day 7 of differentiation, and preincubated in medium with 0, 5, 7.5, or 10 μg/mL BAY11-7082 for 30 minutes at day 6, transfected with a construct containing the −977 to +49 region of the SAR1 gene, then assayed for luciferase activity 24 hours after transfection. * P

    Techniques Used: Binding Assay, Mutagenesis, Isolation, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Amplification, Polymerase Chain Reaction, Transfection, Construct, Luciferase, Activity Assay

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    Immunoprecipitation:

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  • 99
    Thermo Fisher immunoprecipitation buffer
    Biochemical identification of PGRN-interacting proteins. ( A ) mPGRN-AP or mPGRN-HA fusion protein constructs were stably transfected into HEK293 cells. Cell lysates or culture medium was analyzed on Western blot with anti-mPGRN antibody. This antibody specifically recognizes mPGRN but not the endogenous hPGRN in HEK293 cells. β-actin was used as the loading control. ( B ) Stably transfected HEK293 cells expressing mPGRN-HA were treated with cross linkers, followed by <t>immunoprecipitation</t> (IP) with HA antibody or control IgG. Immunoisolates were analyzed on Western blot with anti-mPGRN antibody. UT, untransfected HEK293 cells; ST, stably transfected cells.
    Immunoprecipitation Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher foxp3 staining buffer set
    Reduced in vivo Notch signaling results in reduced <t>Foxp3</t> expression within the T reg pool and spontaneous lymphocyte infiltration of the liver. For 3 months, wild-type mice were fed either normal rodent chow (control, n = 4) or chow formulated with LY 411575 (GSI, n = 5) to deliver 5 mg/kg per day. (A) Bulk splenocytes were stained for flow cytometry with antibodies specific for CD4 and Foxp3. Values represent mean percentages. (B) Histogram representing level of Foxp3 expression, gated on live CD4 + Foxp3 + cells. Data are representative of data collected from 2 independent experimental groups (group 1: control, n = 2 and GSI, n = 3; group 2: control, n = 2 and GSI, n = 2). (C) Graphic representation of mean fluorescence intensity of Foxp3 staining in all experimental groups from panel B. Values represent the mean. (D-G) H E staining of livers from mice with normal (D,F) or reduced (E,G) levels of Notch1 signaling. One representative liver section from each group is shown: (D) control mouse, (E) GSI chow-fed mouse, (F) wild-type mouse (n = 6), (G) Notch1 antisense mouse (n = 6). A Spot digital camera (Diagnostic Instruments, Sterling Heights, MI) mounted on a Zeiss Axioscope microscope (Carl Zeiss, Jena, Germany) using a 20× objective was used to acquire figures.
    Foxp3 Staining Buffer Set, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnase h solution
    RNA detection on the chip with single-nucleotide discrimination through <t>RNase</t> H digestion and Klenow extension at 60°C.
    Rnase H Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biochemical identification of PGRN-interacting proteins. ( A ) mPGRN-AP or mPGRN-HA fusion protein constructs were stably transfected into HEK293 cells. Cell lysates or culture medium was analyzed on Western blot with anti-mPGRN antibody. This antibody specifically recognizes mPGRN but not the endogenous hPGRN in HEK293 cells. β-actin was used as the loading control. ( B ) Stably transfected HEK293 cells expressing mPGRN-HA were treated with cross linkers, followed by immunoprecipitation (IP) with HA antibody or control IgG. Immunoisolates were analyzed on Western blot with anti-mPGRN antibody. UT, untransfected HEK293 cells; ST, stably transfected cells.

    Journal: PLoS ONE

    Article Title: Progranulin, a Glycoprotein Deficient in Frontotemporal Dementia, Is a Novel Substrate of Several Protein Disulfide Isomerase Family Proteins

    doi: 10.1371/journal.pone.0026454

    Figure Lengend Snippet: Biochemical identification of PGRN-interacting proteins. ( A ) mPGRN-AP or mPGRN-HA fusion protein constructs were stably transfected into HEK293 cells. Cell lysates or culture medium was analyzed on Western blot with anti-mPGRN antibody. This antibody specifically recognizes mPGRN but not the endogenous hPGRN in HEK293 cells. β-actin was used as the loading control. ( B ) Stably transfected HEK293 cells expressing mPGRN-HA were treated with cross linkers, followed by immunoprecipitation (IP) with HA antibody or control IgG. Immunoisolates were analyzed on Western blot with anti-mPGRN antibody. UT, untransfected HEK293 cells; ST, stably transfected cells.

    Article Snippet: The cross-link reactions were quenched with 50 mM Tris (pH7.4), and cells were washed with PBS and lysed in immunoprecipitation buffer consisting of 50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 10 mM NaF, 15% glycerol, protease inhibitor cocktail (Pierce), and Halt phosphatase inhibitor cocktail (Pierce).

    Techniques: Construct, Stable Transfection, Transfection, Western Blot, Expressing, Immunoprecipitation

    Biochemical identification of PGRN-interacting proteins. ( A ) Description of the experiment in C . mPGRN-HA stably transfected HEK293 cells were treated with the chemical crosslinker DSS. Immunoprecipitation was performed with an anti-HA antibody. The immunoisolates were analyzed by SDS-PAGE, which was then silver stained. Specific bands were cut out and analyzed by mass spectrometry. ( B ) Image of a gel after silver staining. The identities of bands 1–4 are listed in Table 1 .

    Journal: PLoS ONE

    Article Title: Progranulin, a Glycoprotein Deficient in Frontotemporal Dementia, Is a Novel Substrate of Several Protein Disulfide Isomerase Family Proteins

    doi: 10.1371/journal.pone.0026454

    Figure Lengend Snippet: Biochemical identification of PGRN-interacting proteins. ( A ) Description of the experiment in C . mPGRN-HA stably transfected HEK293 cells were treated with the chemical crosslinker DSS. Immunoprecipitation was performed with an anti-HA antibody. The immunoisolates were analyzed by SDS-PAGE, which was then silver stained. Specific bands were cut out and analyzed by mass spectrometry. ( B ) Image of a gel after silver staining. The identities of bands 1–4 are listed in Table 1 .

    Article Snippet: The cross-link reactions were quenched with 50 mM Tris (pH7.4), and cells were washed with PBS and lysed in immunoprecipitation buffer consisting of 50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 10 mM NaF, 15% glycerol, protease inhibitor cocktail (Pierce), and Halt phosphatase inhibitor cocktail (Pierce).

    Techniques: Stable Transfection, Transfection, Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Silver Staining

    Reduced in vivo Notch signaling results in reduced Foxp3 expression within the T reg pool and spontaneous lymphocyte infiltration of the liver. For 3 months, wild-type mice were fed either normal rodent chow (control, n = 4) or chow formulated with LY 411575 (GSI, n = 5) to deliver 5 mg/kg per day. (A) Bulk splenocytes were stained for flow cytometry with antibodies specific for CD4 and Foxp3. Values represent mean percentages. (B) Histogram representing level of Foxp3 expression, gated on live CD4 + Foxp3 + cells. Data are representative of data collected from 2 independent experimental groups (group 1: control, n = 2 and GSI, n = 3; group 2: control, n = 2 and GSI, n = 2). (C) Graphic representation of mean fluorescence intensity of Foxp3 staining in all experimental groups from panel B. Values represent the mean. (D-G) H E staining of livers from mice with normal (D,F) or reduced (E,G) levels of Notch1 signaling. One representative liver section from each group is shown: (D) control mouse, (E) GSI chow-fed mouse, (F) wild-type mouse (n = 6), (G) Notch1 antisense mouse (n = 6). A Spot digital camera (Diagnostic Instruments, Sterling Heights, MI) mounted on a Zeiss Axioscope microscope (Carl Zeiss, Jena, Germany) using a 20× objective was used to acquire figures.

    Journal: Blood

    Article Title: Notch1 and TGF?1 cooperatively regulate Foxp3 expression and the maintenance of peripheral regulatory T cells

    doi: 10.1182/blood-2008-03-144980

    Figure Lengend Snippet: Reduced in vivo Notch signaling results in reduced Foxp3 expression within the T reg pool and spontaneous lymphocyte infiltration of the liver. For 3 months, wild-type mice were fed either normal rodent chow (control, n = 4) or chow formulated with LY 411575 (GSI, n = 5) to deliver 5 mg/kg per day. (A) Bulk splenocytes were stained for flow cytometry with antibodies specific for CD4 and Foxp3. Values represent mean percentages. (B) Histogram representing level of Foxp3 expression, gated on live CD4 + Foxp3 + cells. Data are representative of data collected from 2 independent experimental groups (group 1: control, n = 2 and GSI, n = 3; group 2: control, n = 2 and GSI, n = 2). (C) Graphic representation of mean fluorescence intensity of Foxp3 staining in all experimental groups from panel B. Values represent the mean. (D-G) H E staining of livers from mice with normal (D,F) or reduced (E,G) levels of Notch1 signaling. One representative liver section from each group is shown: (D) control mouse, (E) GSI chow-fed mouse, (F) wild-type mouse (n = 6), (G) Notch1 antisense mouse (n = 6). A Spot digital camera (Diagnostic Instruments, Sterling Heights, MI) mounted on a Zeiss Axioscope microscope (Carl Zeiss, Jena, Germany) using a 20× objective was used to acquire figures.

    Article Snippet: Intracellular staining for Foxp3 was done using the Foxp3 staining buffer set and anti-Foxp3, clone FJK-16s (both from eBioscience).

    Techniques: In Vivo, Expressing, Mouse Assay, Staining, Flow Cytometry, Cytometry, Fluorescence, Diagnostic Assay, Microscopy

    Impaired Foxp3 induction in cells expressing reduced levels of Notch1. CD4 + CD25 − splenocytes from either wild-type (WT) or Notch1 antisense (AS) mice were stimulated with plate-bound αCD3ϵ plus αCD28 for 72 hours in the presence or absence of 2 ng/mL TGFβ1. (A) Cells were analyzed by flow cytometry using antibodies specific for CD4, CD25, and Foxp3. All plots are gated on live CD4 + cells. Plots are representative of 3 independent experiments. (B) Graphic representation of flow cytometry data from panel A. Data represented as the mean (± SD) of 3 samples per group. * P

    Journal: Blood

    Article Title: Notch1 and TGF?1 cooperatively regulate Foxp3 expression and the maintenance of peripheral regulatory T cells

    doi: 10.1182/blood-2008-03-144980

    Figure Lengend Snippet: Impaired Foxp3 induction in cells expressing reduced levels of Notch1. CD4 + CD25 − splenocytes from either wild-type (WT) or Notch1 antisense (AS) mice were stimulated with plate-bound αCD3ϵ plus αCD28 for 72 hours in the presence or absence of 2 ng/mL TGFβ1. (A) Cells were analyzed by flow cytometry using antibodies specific for CD4, CD25, and Foxp3. All plots are gated on live CD4 + cells. Plots are representative of 3 independent experiments. (B) Graphic representation of flow cytometry data from panel A. Data represented as the mean (± SD) of 3 samples per group. * P

    Article Snippet: Intracellular staining for Foxp3 was done using the Foxp3 staining buffer set and anti-Foxp3, clone FJK-16s (both from eBioscience).

    Techniques: Expressing, Mouse Assay, Flow Cytometry, Cytometry

    In vitro GSI treatment blocks the binding of Notch1, CSL, and Smad to the foxp3 promoter without inhibiting histone acetylation. Chromatin immunoprecipitation (ChIP) of CD4 + CD25 − splenocytes stimulated with plate-bound αCD3ϵ plus αCD28 and 2 ng/mL TGFβ1 in the presence or absence of GSI for 24 hours. (A) Primer sets were designed to span putative CSL and Smad binding sites within the foxp3 promoter. Rabbit αNotch1, rabbit αCSL, and mouse αSmad1/2/3 were used to immunoprecipitate protein-DNA complexes. De–cross-linked DNA was amplified by PCR using either primer set 1 (B) or primer set 2 (D). Band intensities were calculated using ImageJ software, version 1.38 (National Institutes of Health, Bethesda, MD; panels C,E). Data are representative of 3 independent experiments. (F) ChIP assay using antibody specific for acetylated histone H3 (αacetyl H3) and either primer set 1 from panel A or the region of the Foxp3 enhancer containing previously identified Smad3 and NFAT binding sites. Data are representative of 2 independent experiments. Input indicates total chromatin. No Ab (beads only), rabbit IgG, and mouse IgG were used as isotype controls.

    Journal: Blood

    Article Title: Notch1 and TGF?1 cooperatively regulate Foxp3 expression and the maintenance of peripheral regulatory T cells

    doi: 10.1182/blood-2008-03-144980

    Figure Lengend Snippet: In vitro GSI treatment blocks the binding of Notch1, CSL, and Smad to the foxp3 promoter without inhibiting histone acetylation. Chromatin immunoprecipitation (ChIP) of CD4 + CD25 − splenocytes stimulated with plate-bound αCD3ϵ plus αCD28 and 2 ng/mL TGFβ1 in the presence or absence of GSI for 24 hours. (A) Primer sets were designed to span putative CSL and Smad binding sites within the foxp3 promoter. Rabbit αNotch1, rabbit αCSL, and mouse αSmad1/2/3 were used to immunoprecipitate protein-DNA complexes. De–cross-linked DNA was amplified by PCR using either primer set 1 (B) or primer set 2 (D). Band intensities were calculated using ImageJ software, version 1.38 (National Institutes of Health, Bethesda, MD; panels C,E). Data are representative of 3 independent experiments. (F) ChIP assay using antibody specific for acetylated histone H3 (αacetyl H3) and either primer set 1 from panel A or the region of the Foxp3 enhancer containing previously identified Smad3 and NFAT binding sites. Data are representative of 2 independent experiments. Input indicates total chromatin. No Ab (beads only), rabbit IgG, and mouse IgG were used as isotype controls.

    Article Snippet: Intracellular staining for Foxp3 was done using the Foxp3 staining buffer set and anti-Foxp3, clone FJK-16s (both from eBioscience).

    Techniques: In Vitro, Binding Assay, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, Software

    Defective regulatory T-cell maintenance in mice expressing reduced levels of Notch1. Bulk splenocytes (A) or thymocytes (B) were harvested from WT or Notch1 antisense (AS) mice and then stained with antibodies specific for CD4 and Foxp3 for analysis by flow cytometry. * P

    Journal: Blood

    Article Title: Notch1 and TGF?1 cooperatively regulate Foxp3 expression and the maintenance of peripheral regulatory T cells

    doi: 10.1182/blood-2008-03-144980

    Figure Lengend Snippet: Defective regulatory T-cell maintenance in mice expressing reduced levels of Notch1. Bulk splenocytes (A) or thymocytes (B) were harvested from WT or Notch1 antisense (AS) mice and then stained with antibodies specific for CD4 and Foxp3 for analysis by flow cytometry. * P

    Article Snippet: Intracellular staining for Foxp3 was done using the Foxp3 staining buffer set and anti-Foxp3, clone FJK-16s (both from eBioscience).

    Techniques: Mouse Assay, Expressing, Staining, Flow Cytometry, Cytometry

    In vitro GSI treatment blocks TGFβ1-induced Foxp3 expression and function. CD4 + CD25 − splenocytes were isolated and stimulated under the following conditions: no treatment, + TGFβ1, + GSI alone, or + GSI + TGFβ1. (A) Notch1 expression in cells pretreated without or with GSI was evaluated by immunoblotting using antibodies that recognized the cleaved, active form of Notch1 (Notch1 IC ). Antibody specific for HSP70 was used to control for loading. (B) Graphic representation of band intensities shown in panel A. (C) Effect of GSI treatment on Foxp3 expression was analyzed by flow cytometry using antibodies specific for CD4, CD25, and Foxp3. All plots are gated on live CD4 + cells. (D) Graphic representation of flow cytometry data from panel C. Data represent the means (± SD). ** P = .001; * P

    Journal: Blood

    Article Title: Notch1 and TGF?1 cooperatively regulate Foxp3 expression and the maintenance of peripheral regulatory T cells

    doi: 10.1182/blood-2008-03-144980

    Figure Lengend Snippet: In vitro GSI treatment blocks TGFβ1-induced Foxp3 expression and function. CD4 + CD25 − splenocytes were isolated and stimulated under the following conditions: no treatment, + TGFβ1, + GSI alone, or + GSI + TGFβ1. (A) Notch1 expression in cells pretreated without or with GSI was evaluated by immunoblotting using antibodies that recognized the cleaved, active form of Notch1 (Notch1 IC ). Antibody specific for HSP70 was used to control for loading. (B) Graphic representation of band intensities shown in panel A. (C) Effect of GSI treatment on Foxp3 expression was analyzed by flow cytometry using antibodies specific for CD4, CD25, and Foxp3. All plots are gated on live CD4 + cells. (D) Graphic representation of flow cytometry data from panel C. Data represent the means (± SD). ** P = .001; * P

    Article Snippet: Intracellular staining for Foxp3 was done using the Foxp3 staining buffer set and anti-Foxp3, clone FJK-16s (both from eBioscience).

    Techniques: In Vitro, Expressing, Isolation, Flow Cytometry, Cytometry

    BRD9 deletion reduces Foxp3 binding at CNS0, CNS2 enhancers and a subset of Foxp3 target sites A, Genome browser tracks of SMARCA4, BRD9, PHF10 ChIP-seq and ATAC-seq signal, as well as Foxp3 ChIP-seq in sgNT, sgFoxp3, sgBrd9 and sgPbrm1 Tregs and Foxp3 in DMSO and dBRD9 treated Tregs (2.5 μM dBRD9 for 4 days). Foxp3 locus is shown with CNS0 and CNS2 enhancers indicated in gray ovals. B , Heat map of Foxp3, BRD9, SMARCA4, PHF10 ChIP-seq and ATAC-seq signal ± 3 kb centered on Foxp3-bound sites in Tregs, ranked according to Foxp3 read density. C, Venn diagram of the overlap between ChIP-seq peaks in Tregs for BRD9, Foxp3, and PHF10 (hypergeometric p value of BRD9:Foxp3 overlap = e -30704 , hypergeometric p value of PHF10:Foxp3 overlap = e -13182 , hypergeometric p value of BRD9:PHF10 overlap = e -12895 ). D, Heat map of Foxp3 ChIP-seq signal in sgNT, sgFoxp3, sgBrd9 and sgPbrm1 Tregs ± 3 kilobases (kb) centered on Foxp3-bound sites in sgNT, ranked according to read density. E , Venn diagram of the overlap (hypergeometric p value = e -11,653 ) between sites that significantly lose Foxp3 binding (FC 1.5, Poisson p value

    Journal: bioRxiv

    Article Title: A genome-wide CRISPR screen reveals a role for the BRD9-containing non-canonical BAF complex in regulatory T cells

    doi: 10.1101/2020.02.26.964981

    Figure Lengend Snippet: BRD9 deletion reduces Foxp3 binding at CNS0, CNS2 enhancers and a subset of Foxp3 target sites A, Genome browser tracks of SMARCA4, BRD9, PHF10 ChIP-seq and ATAC-seq signal, as well as Foxp3 ChIP-seq in sgNT, sgFoxp3, sgBrd9 and sgPbrm1 Tregs and Foxp3 in DMSO and dBRD9 treated Tregs (2.5 μM dBRD9 for 4 days). Foxp3 locus is shown with CNS0 and CNS2 enhancers indicated in gray ovals. B , Heat map of Foxp3, BRD9, SMARCA4, PHF10 ChIP-seq and ATAC-seq signal ± 3 kb centered on Foxp3-bound sites in Tregs, ranked according to Foxp3 read density. C, Venn diagram of the overlap between ChIP-seq peaks in Tregs for BRD9, Foxp3, and PHF10 (hypergeometric p value of BRD9:Foxp3 overlap = e -30704 , hypergeometric p value of PHF10:Foxp3 overlap = e -13182 , hypergeometric p value of BRD9:PHF10 overlap = e -12895 ). D, Heat map of Foxp3 ChIP-seq signal in sgNT, sgFoxp3, sgBrd9 and sgPbrm1 Tregs ± 3 kilobases (kb) centered on Foxp3-bound sites in sgNT, ranked according to read density. E , Venn diagram of the overlap (hypergeometric p value = e -11,653 ) between sites that significantly lose Foxp3 binding (FC 1.5, Poisson p value

    Article Snippet: Transduced cells were analyzed for Foxp3 and cytokine expression in eBioscience Fix/Perm buffer (eBioscience #00-5523-00) using flow cytometry.

    Techniques: Binding Assay, Chromatin Immunoprecipitation

    BRD9 co-regulates the expression of Foxp3 and a subset of Foxp3 target genes A, Volcano plot of log2 fold change RNA expression in sgFoxp3/sgNT Tregs versus adjusted p value (Benjamin-Hochberg). Number of down- and up-regulated genes are indicated, which are colored blue and red, respectively. B, Significance of enrichment of Foxp3-dependent genes in each gene ontology. C, Pie chart of Foxp3 and BRD9 binding by ChIP-seq for Foxp3-dependent genes. D, Scatterplot of the mRNA log2 fold changes in sgFoxp3/sgNT and sgBrd9/sgNT for Foxp3-dependent genes. Linear regression analysis was performed to calculate the r 2 . Best fit is represented as an orange dashed line. E, Gene set enrichment analysis (GSEA) enrichment plot for up- and down-regulated genes in sgBrd9/sgNT compared with RNA-seq data of genes that significantly change in sgFoxp3/sgNT Tregs. ES: Enrichment Score, NES: Normalized Enrichment Score, FWER: Familywise Error Rate. F , As in E , but for up- and down-regulated genes in dBRD9/DMSO Tregs. G, GSEA of the sgFoxp3/sgNT RNA-seq data; plot shows the familywise error rate (FWER) p value versus the normalized enrichment score (NES). See also Table S5.

    Journal: bioRxiv

    Article Title: A genome-wide CRISPR screen reveals a role for the BRD9-containing non-canonical BAF complex in regulatory T cells

    doi: 10.1101/2020.02.26.964981

    Figure Lengend Snippet: BRD9 co-regulates the expression of Foxp3 and a subset of Foxp3 target genes A, Volcano plot of log2 fold change RNA expression in sgFoxp3/sgNT Tregs versus adjusted p value (Benjamin-Hochberg). Number of down- and up-regulated genes are indicated, which are colored blue and red, respectively. B, Significance of enrichment of Foxp3-dependent genes in each gene ontology. C, Pie chart of Foxp3 and BRD9 binding by ChIP-seq for Foxp3-dependent genes. D, Scatterplot of the mRNA log2 fold changes in sgFoxp3/sgNT and sgBrd9/sgNT for Foxp3-dependent genes. Linear regression analysis was performed to calculate the r 2 . Best fit is represented as an orange dashed line. E, Gene set enrichment analysis (GSEA) enrichment plot for up- and down-regulated genes in sgBrd9/sgNT compared with RNA-seq data of genes that significantly change in sgFoxp3/sgNT Tregs. ES: Enrichment Score, NES: Normalized Enrichment Score, FWER: Familywise Error Rate. F , As in E , but for up- and down-regulated genes in dBRD9/DMSO Tregs. G, GSEA of the sgFoxp3/sgNT RNA-seq data; plot shows the familywise error rate (FWER) p value versus the normalized enrichment score (NES). See also Table S5.

    Article Snippet: Transduced cells were analyzed for Foxp3 and cytokine expression in eBioscience Fix/Perm buffer (eBioscience #00-5523-00) using flow cytometry.

    Techniques: Expressing, RNA Expression, Binding Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay

    Identification of novel Foxp3 regulators in Treg cells A, B , A scatter plot of the Treg screen result showing positive regulators (A) and negative regulators (B). Genes that have met cutoff criteria (P-value

    Journal: bioRxiv

    Article Title: A genome-wide CRISPR screen reveals a role for the BRD9-containing non-canonical BAF complex in regulatory T cells

    doi: 10.1101/2020.02.26.964981

    Figure Lengend Snippet: Identification of novel Foxp3 regulators in Treg cells A, B , A scatter plot of the Treg screen result showing positive regulators (A) and negative regulators (B). Genes that have met cutoff criteria (P-value

    Article Snippet: Transduced cells were analyzed for Foxp3 and cytokine expression in eBioscience Fix/Perm buffer (eBioscience #00-5523-00) using flow cytometry.

    Techniques: Significance Assay

    Targeting BRD9 in Treg improves anti-tumor immunity. A, Experiment procedure to measure function of sgNT or sgBrd9 knockout Treg cells relative to no Tregs in MC38 tumor model. B , Tumor growth curve. C , Tumor weight at end point. D,E , Bar graph of total CD4 T cells ( D ) and CD8 T cells ( E ) percentage in CD45+ immune cell population. F,G , Bar graph of IFN-γ+ cell percentage in CD4 T cells ( F ) and in CD8 T cells ( G ). H , Bar graph of CD4+eGFP+Foxp3+ donor cells in CD4+ T cells . I, Ratio of CD8/Treg. (n=5-7 per group. Data represent mean ± s.e.m.) Statistical analyses were performed using unpaired two-tailed Student’s t test (ns: p≥0.05, *p

    Journal: bioRxiv

    Article Title: A genome-wide CRISPR screen reveals a role for the BRD9-containing non-canonical BAF complex in regulatory T cells

    doi: 10.1101/2020.02.26.964981

    Figure Lengend Snippet: Targeting BRD9 in Treg improves anti-tumor immunity. A, Experiment procedure to measure function of sgNT or sgBrd9 knockout Treg cells relative to no Tregs in MC38 tumor model. B , Tumor growth curve. C , Tumor weight at end point. D,E , Bar graph of total CD4 T cells ( D ) and CD8 T cells ( E ) percentage in CD45+ immune cell population. F,G , Bar graph of IFN-γ+ cell percentage in CD4 T cells ( F ) and in CD8 T cells ( G ). H , Bar graph of CD4+eGFP+Foxp3+ donor cells in CD4+ T cells . I, Ratio of CD8/Treg. (n=5-7 per group. Data represent mean ± s.e.m.) Statistical analyses were performed using unpaired two-tailed Student’s t test (ns: p≥0.05, *p

    Article Snippet: Transduced cells were analyzed for Foxp3 and cytokine expression in eBioscience Fix/Perm buffer (eBioscience #00-5523-00) using flow cytometry.

    Techniques: Knock-Out, Two Tailed Test

    The three SWI/SNF complex assemblies have distinct regulatory roles for Foxp3 expression in Tregs A, A diagram showing three different variants of SWI/SNF complexes: BAF, ncBAF, and PBAF. BAF-specific subunits (ARID1A, DPF1-3) are colored blue, ncBAF-specific subunits (BRD9, SMARCD1, GLTSCR1L, GLTSCR1) colored orange, and PBAF-specific subunit (PBRM1, ARID2, BRD7, PHF10) colored green. Shared components among complexes are colored gray. Immunoprecipitation assay of ARID1A, BRD9, and PHF10, and BRG1 in Tregs. The co-precipitated proteins were probed for shared subunits (SMARCA4, SMARCC1, SMARCB1), BAF-specific ARID1A, ncBAF-specific BRD9, and PBAF-specific PBRM1. B, FACS histogram of Foxp3 expression in Tregs after sgRNA knockout of the indicated SWI/SNF subunits. C, Mean fluorescence intensity (MFI) of Foxp3 after sgRNA knockout of the indicated SWI/SNF subunits. Data represents mean and standard deviation of biological replicates (n = 3-21). D, Principal component analysis of RNA-seq data collected from Tregs transduced with guides against the indicated SWI/SNF subunits. In cases where two independent guides were used to knockdown a gene, the second guide for targeting gene indicated as “-2”. E, MFI of Foxp3 expression in Tregs after treatment with either DMSO or 0.16-10 μM dBRD9 for 4 days. Data represent mean ± s.d. Statistical analyses were performed using unpaired two-tailed Student’s t test (ns: p≥0.05, *p

    Journal: bioRxiv

    Article Title: A genome-wide CRISPR screen reveals a role for the BRD9-containing non-canonical BAF complex in regulatory T cells

    doi: 10.1101/2020.02.26.964981

    Figure Lengend Snippet: The three SWI/SNF complex assemblies have distinct regulatory roles for Foxp3 expression in Tregs A, A diagram showing three different variants of SWI/SNF complexes: BAF, ncBAF, and PBAF. BAF-specific subunits (ARID1A, DPF1-3) are colored blue, ncBAF-specific subunits (BRD9, SMARCD1, GLTSCR1L, GLTSCR1) colored orange, and PBAF-specific subunit (PBRM1, ARID2, BRD7, PHF10) colored green. Shared components among complexes are colored gray. Immunoprecipitation assay of ARID1A, BRD9, and PHF10, and BRG1 in Tregs. The co-precipitated proteins were probed for shared subunits (SMARCA4, SMARCC1, SMARCB1), BAF-specific ARID1A, ncBAF-specific BRD9, and PBAF-specific PBRM1. B, FACS histogram of Foxp3 expression in Tregs after sgRNA knockout of the indicated SWI/SNF subunits. C, Mean fluorescence intensity (MFI) of Foxp3 after sgRNA knockout of the indicated SWI/SNF subunits. Data represents mean and standard deviation of biological replicates (n = 3-21). D, Principal component analysis of RNA-seq data collected from Tregs transduced with guides against the indicated SWI/SNF subunits. In cases where two independent guides were used to knockdown a gene, the second guide for targeting gene indicated as “-2”. E, MFI of Foxp3 expression in Tregs after treatment with either DMSO or 0.16-10 μM dBRD9 for 4 days. Data represent mean ± s.d. Statistical analyses were performed using unpaired two-tailed Student’s t test (ns: p≥0.05, *p

    Article Snippet: Transduced cells were analyzed for Foxp3 and cytokine expression in eBioscience Fix/Perm buffer (eBioscience #00-5523-00) using flow cytometry.

    Techniques: Expressing, Immunoprecipitation, FACS, Knock-Out, Fluorescence, Standard Deviation, RNA Sequencing Assay, Transduction, Two Tailed Test

    A genome-wide CRISPR screen in Treg cells A, Workflow of the CRISPR screen in Tregs. B-D, Validation of the CRISPR screen conditions. B, FACS plots showing Foxp3 expression in Tregs after knocking out Foxp3 (sgFoxp3), positive regulator Cbfb (sgCbfb), and negative regulator Dnmt1 (sgDnmt1). Red and green gates were set based on Foxp3 low 20% and high 20% in sgNT Treg, respectively. C, Mean fluorescence intensity (MFI) of Foxp3 and D, Relative Log2FC of cell count comparing Foxp3 Low to Foxp3 High after deletion of the indicated target gene (n=3 per group). See also Figure S1 and S2.

    Journal: bioRxiv

    Article Title: A genome-wide CRISPR screen reveals a role for the BRD9-containing non-canonical BAF complex in regulatory T cells

    doi: 10.1101/2020.02.26.964981

    Figure Lengend Snippet: A genome-wide CRISPR screen in Treg cells A, Workflow of the CRISPR screen in Tregs. B-D, Validation of the CRISPR screen conditions. B, FACS plots showing Foxp3 expression in Tregs after knocking out Foxp3 (sgFoxp3), positive regulator Cbfb (sgCbfb), and negative regulator Dnmt1 (sgDnmt1). Red and green gates were set based on Foxp3 low 20% and high 20% in sgNT Treg, respectively. C, Mean fluorescence intensity (MFI) of Foxp3 and D, Relative Log2FC of cell count comparing Foxp3 Low to Foxp3 High after deletion of the indicated target gene (n=3 per group). See also Figure S1 and S2.

    Article Snippet: Transduced cells were analyzed for Foxp3 and cytokine expression in eBioscience Fix/Perm buffer (eBioscience #00-5523-00) using flow cytometry.

    Techniques: Genome Wide, CRISPR, FACS, Expressing, Fluorescence, Cell Counting

    The ncBAF complex regulates Treg suppressor function in vitro and in vivo. A. In vitro suppression assay of Tregs with sgRNA knockout of Brd9, Smarcd1, Pbrm1, and Phf10 (n=3 per group, data represent ± s.d.). sgNT was used as non-targeting control. B-F. Experiment to measure Treg function of sgNT or sgBrd9 knockout Treg cells relative to no Tregs in a T cell transfer induced colitis model. B, Experimental procedure. C, Body weight loss. D, Colon histology (left) and colitis scores (right). E, Percentage of Foxp3+ cells in transferred CD45.2+CD4+ Treg population at end point. (n=4-6 per group. Data represent mean ± s.e.m.) Statistical analyses were performed using unpaired two-tailed Student’s t test (ns: p≥0.05, *p

    Journal: bioRxiv

    Article Title: A genome-wide CRISPR screen reveals a role for the BRD9-containing non-canonical BAF complex in regulatory T cells

    doi: 10.1101/2020.02.26.964981

    Figure Lengend Snippet: The ncBAF complex regulates Treg suppressor function in vitro and in vivo. A. In vitro suppression assay of Tregs with sgRNA knockout of Brd9, Smarcd1, Pbrm1, and Phf10 (n=3 per group, data represent ± s.d.). sgNT was used as non-targeting control. B-F. Experiment to measure Treg function of sgNT or sgBrd9 knockout Treg cells relative to no Tregs in a T cell transfer induced colitis model. B, Experimental procedure. C, Body weight loss. D, Colon histology (left) and colitis scores (right). E, Percentage of Foxp3+ cells in transferred CD45.2+CD4+ Treg population at end point. (n=4-6 per group. Data represent mean ± s.e.m.) Statistical analyses were performed using unpaired two-tailed Student’s t test (ns: p≥0.05, *p

    Article Snippet: Transduced cells were analyzed for Foxp3 and cytokine expression in eBioscience Fix/Perm buffer (eBioscience #00-5523-00) using flow cytometry.

    Techniques: In Vitro, In Vivo, Suppression Assay, Knock-Out, Two Tailed Test

    RNA detection on the chip with single-nucleotide discrimination through RNase H digestion and Klenow extension at 60°C.

    Journal: Chembiochem : a European journal of chemical biology

    Article Title: Direct and Rapid Detection of RNAs on a Novel RNA Microchip

    doi: 10.1002/cbic.201000170

    Figure Lengend Snippet: RNA detection on the chip with single-nucleotide discrimination through RNase H digestion and Klenow extension at 60°C.

    Article Snippet: The microchip was then incubated with RNase H buffer (20 μL, 1×, New England Biolabs) and RNase H (0.5 μL, 5000 U mL−1 , New England Biolabs) at 37°C for 5 min. After the RNase H solution had been drained from the incubation chamber, the microchip surface was quickly rinsed with StartingBlock (TBS) blocking buffer (Pierce).

    Techniques: RNA Detection, Chromatin Immunoprecipitation