Journal: mAbs

Article Title: The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

doi: 10.1080/19420862.2015.1008353

Figure Lengend Snippet: Affinity determination of the rFcRn/rIgG2a interaction at pH 5.8 using various assay orientations. Kinetic analysis of rIgG2a flowed over biotinylated rFcRn that was captured at ( A ) high or ( B ) low capacities via amine-coupled neutravidin on a Biacore C1 chip; kinetic analysis of rFcRn flowed over ( C ) antigen-captured rIgG2a on a ProteOn NLC chip or ( D ) amine-coupled rIgG2a on a Biacore CM5 chip; and ( E ) solution affinity using a high capacity of amine-coupled rIgG2a on a Biacore CM5 chip to probe for free rFcRn in equilibrated mixtures with titrating levels of rIgG2a. Analytes were injected as a 3-fold dilution series with top at 500 nM ( A and B ) or as both a 3-fold (green) and 5-fold (red) dilution series with top at 1000 nM ( C ) or 3000 nM ( D ); the 3000 nM curves have been excluded from D . Panel E shows the titration of 17 nM rFcRn with 2 unrelated mAbs of subtype rIgG2a (distinguished by the solid or open symbols). All samples were analyzed in replicate binding cycles. Each panel shows an example data set (N of 1) where the measured data (colored lines) were fit globally to a simple model (black lines). The K D values are the mean ± SD for N independent measurements. See Table 1 .

Article Snippet: Within a given assay format, such as FcRn kinetics on antigen-captured IgGs, the apparent affinities determined on ProteOn's NLC, GLC, and GLM chips and Biacore's C1 chip agreed more closely with solution measurements than those obtained on Biacore's SA, CM4, or CM5 chips, which yielded weaker affinities driven by slower apparent association rates in these chip types, as noted in the literature for other interaction systems.

Techniques: Injection, Titration, Binding Assay

Journal: mAbs

Article Title: The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

doi: 10.1080/19420862.2015.1008353

Figure Lengend Snippet: Affinity determination of the rFcRn/rIgG2a interaction at pH 5.8 using 3 different assay formats on SPR platforms. Parameter values represent the mean ± SD of N independent measurements on 5 unrelated mAbs. The range of K D values obtained is also shown in parenthesis. ND = not determined

Article Snippet: Within a given assay format, such as FcRn kinetics on antigen-captured IgGs, the apparent affinities determined on ProteOn's NLC, GLC, and GLM chips and Biacore's C1 chip agreed more closely with solution measurements than those obtained on Biacore's SA, CM4, or CM5 chips, which yielded weaker affinities driven by slower apparent association rates in these chip types, as noted in the literature for other interaction systems.

Techniques:

Journal: mAbs

Article Title: The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

doi: 10.1080/19420862.2015.1008353

Figure Lengend Snippet: Kinetic analysis at pH 5.8 of a multi-species panel of FcRn proteins binding as analytes to trastuzumab hIgG1-N434Y that was first captured via ( A ) biotinylated mFcRn or ( B ) biotinylated hErbB2 on Biacore C1 chips to which neutravidin was amine-coupled. Analytes were injected in duplicate binding cycles as a 3-fold dilution series with top at 180, 300, 60, or 100 nM for hFcRn, cyFcRn, mFcRn, and rFcRn respectively. Each overlay plot shows a representative example of the measured data (colored lines) and the global fit to a simple model (black lines) for a typical experiment (N of 1) and the K D values are the mean ± SD of N = 6.

Article Snippet: Within a given assay format, such as FcRn kinetics on antigen-captured IgGs, the apparent affinities determined on ProteOn's NLC, GLC, and GLM chips and Biacore's C1 chip agreed more closely with solution measurements than those obtained on Biacore's SA, CM4, or CM5 chips, which yielded weaker affinities driven by slower apparent association rates in these chip types, as noted in the literature for other interaction systems.

Techniques: Binding Assay, Injection

Journal: mAbs

Article Title: The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

doi: 10.1080/19420862.2015.1008353

Figure Lengend Snippet: Affinity determination of the rFcRn/rIgG2a interaction at pH 5.8 using various assay orientations. Kinetic analysis of rIgG2a flowed over biotinylated rFcRn that was captured at ( A ) high or ( B ) low capacities via amine-coupled neutravidin on a Biacore C1 chip; kinetic analysis of rFcRn flowed over ( C ) antigen-captured rIgG2a on a ProteOn NLC chip or ( D ) amine-coupled rIgG2a on a Biacore CM5 chip; and ( E ) solution affinity using a high capacity of amine-coupled rIgG2a on a Biacore CM5 chip to probe for free rFcRn in equilibrated mixtures with titrating levels of rIgG2a. Analytes were injected as a 3-fold dilution series with top at 500 nM ( A and B ) or as both a 3-fold (green) and 5-fold (red) dilution series with top at 1000 nM ( C ) or 3000 nM ( D ); the 3000 nM curves have been excluded from D . Panel E shows the titration of 17 nM rFcRn with 2 unrelated mAbs of subtype rIgG2a (distinguished by the solid or open symbols). All samples were analyzed in replicate binding cycles. Each panel shows an example data set (N of 1) where the measured data (colored lines) were fit globally to a simple model (black lines). The K D values are the mean ± SD for N independent measurements. See Table 1 .

Article Snippet: Kinetic analysis of rIgG2a flowed over biotinylated rFcRn that was captured at ( A ) high or ( B ) low capacities via amine-coupled neutravidin on a Biacore C1 chip; kinetic analysis of rFcRn flowed over ( C ) antigen-captured rIgG2a on a ProteOn NLC chip or ( D ) amine-coupled rIgG2a on a Biacore CM5 chip; and ( E ) solution affinity using a high capacity of amine-coupled rIgG2a on a Biacore CM5 chip to probe for free rFcRn in equilibrated mixtures with titrating levels of rIgG2a.

Techniques: Injection, Titration, Binding Assay

Journal: mAbs

Article Title: The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

doi: 10.1080/19420862.2015.1008353

Figure Lengend Snippet: Affinity determination of the rFcRn/rIgG2a interaction at pH 5.8 using 3 different assay formats on SPR platforms. Parameter values represent the mean ± SD of N independent measurements on 5 unrelated mAbs. The range of K D values obtained is also shown in parenthesis. ND = not determined

Article Snippet: Kinetic analysis of rIgG2a flowed over biotinylated rFcRn that was captured at ( A ) high or ( B ) low capacities via amine-coupled neutravidin on a Biacore C1 chip; kinetic analysis of rFcRn flowed over ( C ) antigen-captured rIgG2a on a ProteOn NLC chip or ( D ) amine-coupled rIgG2a on a Biacore CM5 chip; and ( E ) solution affinity using a high capacity of amine-coupled rIgG2a on a Biacore CM5 chip to probe for free rFcRn in equilibrated mixtures with titrating levels of rIgG2a.

Techniques:

Journal: mAbs

Article Title: The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

doi: 10.1080/19420862.2015.1008353

Figure Lengend Snippet: Kinetic analysis at pH 5.8 of a multi-species panel of FcRn proteins binding as analytes to trastuzumab hIgG1-N434Y that was first captured via ( A ) biotinylated mFcRn or ( B ) biotinylated hErbB2 on Biacore C1 chips to which neutravidin was amine-coupled. Analytes were injected in duplicate binding cycles as a 3-fold dilution series with top at 180, 300, 60, or 100 nM for hFcRn, cyFcRn, mFcRn, and rFcRn respectively. Each overlay plot shows a representative example of the measured data (colored lines) and the global fit to a simple model (black lines) for a typical experiment (N of 1) and the K D values are the mean ± SD of N = 6.

Article Snippet: Kinetic analysis of rIgG2a flowed over biotinylated rFcRn that was captured at ( A ) high or ( B ) low capacities via amine-coupled neutravidin on a Biacore C1 chip; kinetic analysis of rFcRn flowed over ( C ) antigen-captured rIgG2a on a ProteOn NLC chip or ( D ) amine-coupled rIgG2a on a Biacore CM5 chip; and ( E ) solution affinity using a high capacity of amine-coupled rIgG2a on a Biacore CM5 chip to probe for free rFcRn in equilibrated mixtures with titrating levels of rIgG2a.

Techniques: Binding Assay, Injection

Journal: mAbs

Article Title: The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

doi: 10.1080/19420862.2015.1008353

Figure Lengend Snippet: Affinity determination of the rFcRn/rIgG2a interaction at pH 5.8 using various assay orientations. Kinetic analysis of rIgG2a flowed over biotinylated rFcRn that was captured at ( A ) high or ( B ) low capacities via amine-coupled neutravidin on a Biacore C1 chip; kinetic analysis of rFcRn flowed over ( C ) antigen-captured rIgG2a on a ProteOn NLC chip or ( D ) amine-coupled rIgG2a on a Biacore CM5 chip; and ( E ) solution affinity using a high capacity of amine-coupled rIgG2a on a Biacore CM5 chip to probe for free rFcRn in equilibrated mixtures with titrating levels of rIgG2a. Analytes were injected as a 3-fold dilution series with top at 500 nM ( A and B ) or as both a 3-fold (green) and 5-fold (red) dilution series with top at 1000 nM ( C ) or 3000 nM ( D ); the 3000 nM curves have been excluded from D . Panel E shows the titration of 17 nM rFcRn with 2 unrelated mAbs of subtype rIgG2a (distinguished by the solid or open symbols). All samples were analyzed in replicate binding cycles. Each panel shows an example data set (N of 1) where the measured data (colored lines) were fit globally to a simple model (black lines). The K D values are the mean ± SD for N independent measurements. See Table 1 .

Article Snippet: Within a given assay format, such as FcRn kinetics on antigen-captured IgGs, the apparent affinities determined on ProteOn's NLC, GLC, and GLM chips and Biacore's C1 chip agreed more closely with solution measurements than those obtained on Biacore's SA, CM4, or CM5 chips, which yielded weaker affinities driven by slower apparent association rates in these chip types, as noted in the literature for other interaction systems.

Techniques: Injection, Titration, Binding Assay

Journal: mAbs

Article Title: The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

doi: 10.1080/19420862.2015.1008353

Figure Lengend Snippet: Affinity determination of the rFcRn/rIgG2a interaction at pH 5.8 using 3 different assay formats on SPR platforms. Parameter values represent the mean ± SD of N independent measurements on 5 unrelated mAbs. The range of K D values obtained is also shown in parenthesis. ND = not determined

Article Snippet: Within a given assay format, such as FcRn kinetics on antigen-captured IgGs, the apparent affinities determined on ProteOn's NLC, GLC, and GLM chips and Biacore's C1 chip agreed more closely with solution measurements than those obtained on Biacore's SA, CM4, or CM5 chips, which yielded weaker affinities driven by slower apparent association rates in these chip types, as noted in the literature for other interaction systems.

Techniques:

Journal:

Article Title: Restricted V gene usage and VH/VL pairing of mouse humoral response against the N-terminal immunodominant epitope of the amyloid ? peptide

doi: 10.1016/j.molimm.2010.09.012

Figure Lengend Snippet: Kinetic constants and affinities of various immobilized rFab with Aβ 1–16 -Im7.

Article Snippet: Two different assay formats were designed to analyze the binding of Fab to Aβ: (1) Capture Assay : recombinant Aβ 1–16 -Im7 fusion protein, used as a “surrogate Aβ antigen” (for details, see Section 3), was injected over Fab captured on the surface of a GLC chip containing previously immobilized Human Fab Binder (GE Healthcare); and (2) Direct Assay : recombinant Fabs were injected over various Aβ forms captured on a NeutrAvidin coated chip (NLC, BioRad).

Techniques: Mutagenesis

Journal: Cell reports

Article Title: The mTOR chromatin-bound interactome in prostate cancer.

doi: 10.1016/j.celrep.2022.110534

Figure Lengend Snippet: Figure 1. RIME identification of mTOR CIPs in PCa cells (A) Schematic of mTOR RIME analysis in four PCa models treated with vehicle (DMSO), the synthetic androgen R1881, and/or mTOR inhibitor Torin 1 in biological triplicates. Vehicle-treated immunoglobulin G controls were also used. (B) mTOR protein structure and cumulative peptide coverage from mTOR RIME analysis across triplicate experiments in four PCa cell lines. (C) Total mTOR RIME CIPs identified and whether they were previously known. (D) Immunoblot analysis of mTOR, AR full-length (AR-FL) and splice variant (AR-V7), and PTEN levels in nuclear PCa homogenates. Lamin B1 levels are shown as a loading control. (E) Overlap of mTOR RIME datasets between PCa cell lines/conditions. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER ON-TARGETplus SMARTpool non-targeting control siRNAs Dharmacon (Horizon Discovery) Cat# D-001810-10-20 Human shHDAC2#1: CAGTCTCACCAATTT CAGAAA Sigma-Aldrich Cat # TRCN0000004819 Human shHDAC2#2: GCCTATTATCTCAAA GGTGAT Sigma-Aldrich Cat# TRCN0000004821 Human gene-specific primers for qRT-PCR analysis This paper Table S7 Human gene-specific primers for ChIP-qPCR analysis This paper Table S7 Recombinant DNA ViraPowerTM Lentiviral Packaging Mix Thermo Fisher Scientific Cat# K4975-00 Lentiviral packaging plasmid psPAX2 addgene Cat# 12260 VSV-G envelope expressing plasmid pMD2.G addgene Cat# 12259 mTOR shRNA plasmid addgene Cat# 1856 Software and algorithms Proteome Discoverer v2.3 Thermo Scientific Cat# OPTON-20141 Mascot v2.6 Matrix Science http://www.matrixscience.com/ Scaffold v4.10.0 Proteome Software https://www.proteomesoftware.com/ Phantasus v1.11.0 Bioconductor (https://doi.org/ 10.18129/B9.bioc.phantasus) https://genome.ifmo.ru/phantasus ExPASy PeptideCutter Duvaud et al., 2021 https://web.expasy.org/peptide_cutter/ WebGestalt Liao et al., 2019 RRID:SCR_006786 Ingenuity Pathway Analysis (IPA) QIAGEN RRID:SCR_008653 Metascape Zhou et al., 2019 RRID:SCR_016620 Intervene Khan and Mathelier, 2017 https://asntech.shinyapps.io/intervene/ ChIP-Atlas Oki et al., 2018 RRID:SCR_015511 HOMER v4.11 Heinz et al., 2010 RRID:SCR_010881 BioGRID v3.5 Oughtred et al., 2019 RRID:SCR_007393 STRING v11.0 Szklarczyk et al., 2019 RRID:SCR_005223 HuRI Luck et al., 2020 RRID:SCR_015670 IID Kotlyar et al., 2016 http://ophid.utoronto.ca/iid Morpheus Broad Institute RRID:SCR_017386 Cancer Dependency Map (DepMap) portal Broad Institute RRID:SCR_017655 Prism v9 GraphPad https://www.graphpad.com/ scientific-software/prism/ Other Easy-nLC II system Thermo Fisher Scientific (Proxeon Biosystems) https://www.thermofisher.com/ LTQ Orbitrap Velos spectrometer Thermo Fisher Scientific https://www.thermofisher.com/ ChemiDoc MP imaging system Bio-Rad Cat# 12003154 LightCycler 480 system Roche https://diagnostics.roche.com/ global/en/products/systems/lightcycler480-system.html

Techniques: Western Blot, Variant Assay, Control

Journal: Cell reports

Article Title: The mTOR chromatin-bound interactome in prostate cancer.

doi: 10.1016/j.celrep.2022.110534

Figure Lengend Snippet: Figure 2. Effect of R1881 and/or Torin 1 on chromatin mTOR-protein interactions (A) Overlap of drug-modulated mTOR CIPs from triplicate RIME experiments in AR+ LNCaP and 22Rv1 cells. (B) R1881-modulated chromatin-bound mTOR-protein affinities. Gene names for a subset of the CIPs are shown. Red, enhanced/gained interactions; blue, reduced/lost interactions. (C) AR protein structure and cumulative peptide coverage from mTOR RIME analysis across triplicate experiments in AR+ cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER ON-TARGETplus SMARTpool non-targeting control siRNAs Dharmacon (Horizon Discovery) Cat# D-001810-10-20 Human shHDAC2#1: CAGTCTCACCAATTT CAGAAA Sigma-Aldrich Cat # TRCN0000004819 Human shHDAC2#2: GCCTATTATCTCAAA GGTGAT Sigma-Aldrich Cat# TRCN0000004821 Human gene-specific primers for qRT-PCR analysis This paper Table S7 Human gene-specific primers for ChIP-qPCR analysis This paper Table S7 Recombinant DNA ViraPowerTM Lentiviral Packaging Mix Thermo Fisher Scientific Cat# K4975-00 Lentiviral packaging plasmid psPAX2 addgene Cat# 12260 VSV-G envelope expressing plasmid pMD2.G addgene Cat# 12259 mTOR shRNA plasmid addgene Cat# 1856 Software and algorithms Proteome Discoverer v2.3 Thermo Scientific Cat# OPTON-20141 Mascot v2.6 Matrix Science http://www.matrixscience.com/ Scaffold v4.10.0 Proteome Software https://www.proteomesoftware.com/ Phantasus v1.11.0 Bioconductor (https://doi.org/ 10.18129/B9.bioc.phantasus) https://genome.ifmo.ru/phantasus ExPASy PeptideCutter Duvaud et al., 2021 https://web.expasy.org/peptide_cutter/ WebGestalt Liao et al., 2019 RRID:SCR_006786 Ingenuity Pathway Analysis (IPA) QIAGEN RRID:SCR_008653 Metascape Zhou et al., 2019 RRID:SCR_016620 Intervene Khan and Mathelier, 2017 https://asntech.shinyapps.io/intervene/ ChIP-Atlas Oki et al., 2018 RRID:SCR_015511 HOMER v4.11 Heinz et al., 2010 RRID:SCR_010881 BioGRID v3.5 Oughtred et al., 2019 RRID:SCR_007393 STRING v11.0 Szklarczyk et al., 2019 RRID:SCR_005223 HuRI Luck et al., 2020 RRID:SCR_015670 IID Kotlyar et al., 2016 http://ophid.utoronto.ca/iid Morpheus Broad Institute RRID:SCR_017386 Cancer Dependency Map (DepMap) portal Broad Institute RRID:SCR_017655 Prism v9 GraphPad https://www.graphpad.com/ scientific-software/prism/ Other Easy-nLC II system Thermo Fisher Scientific (Proxeon Biosystems) https://www.thermofisher.com/ LTQ Orbitrap Velos spectrometer Thermo Fisher Scientific https://www.thermofisher.com/ ChemiDoc MP imaging system Bio-Rad Cat# 12003154 LightCycler 480 system Roche https://diagnostics.roche.com/ global/en/products/systems/lightcycler480-system.html

Techniques:

Journal: Cell reports

Article Title: The mTOR chromatin-bound interactome in prostate cancer.

doi: 10.1016/j.celrep.2022.110534

Figure Lengend Snippet: Figure 3. Functional enrichment analysis of mTOR CIPs in LNCaP cells ± R1881 (A) Venn diagrams showing the number of mTOR CIPs identified in LNCaP cells from biological triplicates under vehicle or R1881 conditions versus immuno- globulin G control with 87% of the total having a known nuclear localization.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER ON-TARGETplus SMARTpool non-targeting control siRNAs Dharmacon (Horizon Discovery) Cat# D-001810-10-20 Human shHDAC2#1: CAGTCTCACCAATTT CAGAAA Sigma-Aldrich Cat # TRCN0000004819 Human shHDAC2#2: GCCTATTATCTCAAA GGTGAT Sigma-Aldrich Cat# TRCN0000004821 Human gene-specific primers for qRT-PCR analysis This paper Table S7 Human gene-specific primers for ChIP-qPCR analysis This paper Table S7 Recombinant DNA ViraPowerTM Lentiviral Packaging Mix Thermo Fisher Scientific Cat# K4975-00 Lentiviral packaging plasmid psPAX2 addgene Cat# 12260 VSV-G envelope expressing plasmid pMD2.G addgene Cat# 12259 mTOR shRNA plasmid addgene Cat# 1856 Software and algorithms Proteome Discoverer v2.3 Thermo Scientific Cat# OPTON-20141 Mascot v2.6 Matrix Science http://www.matrixscience.com/ Scaffold v4.10.0 Proteome Software https://www.proteomesoftware.com/ Phantasus v1.11.0 Bioconductor (https://doi.org/ 10.18129/B9.bioc.phantasus) https://genome.ifmo.ru/phantasus ExPASy PeptideCutter Duvaud et al., 2021 https://web.expasy.org/peptide_cutter/ WebGestalt Liao et al., 2019 RRID:SCR_006786 Ingenuity Pathway Analysis (IPA) QIAGEN RRID:SCR_008653 Metascape Zhou et al., 2019 RRID:SCR_016620 Intervene Khan and Mathelier, 2017 https://asntech.shinyapps.io/intervene/ ChIP-Atlas Oki et al., 2018 RRID:SCR_015511 HOMER v4.11 Heinz et al., 2010 RRID:SCR_010881 BioGRID v3.5 Oughtred et al., 2019 RRID:SCR_007393 STRING v11.0 Szklarczyk et al., 2019 RRID:SCR_005223 HuRI Luck et al., 2020 RRID:SCR_015670 IID Kotlyar et al., 2016 http://ophid.utoronto.ca/iid Morpheus Broad Institute RRID:SCR_017386 Cancer Dependency Map (DepMap) portal Broad Institute RRID:SCR_017655 Prism v9 GraphPad https://www.graphpad.com/ scientific-software/prism/ Other Easy-nLC II system Thermo Fisher Scientific (Proxeon Biosystems) https://www.thermofisher.com/ LTQ Orbitrap Velos spectrometer Thermo Fisher Scientific https://www.thermofisher.com/ ChemiDoc MP imaging system Bio-Rad Cat# 12003154 LightCycler 480 system Roche https://diagnostics.roche.com/ global/en/products/systems/lightcycler480-system.html

Techniques: Functional Assay, Control

Journal: Cell reports

Article Title: The mTOR chromatin-bound interactome in prostate cancer.

doi: 10.1016/j.celrep.2022.110534

Figure Lengend Snippet: Figure 4. Identification of a conserved mTOR chromatin network in PCa cells (A) Left, common significantly over-represented Ingenuity Pathway Analysis (IPA) canonical pathways from mTOR RIME analysis in four PCa cell lines under basal conditions (vehicle-treated) performed in biological triplicates. Right, mTOR RIME associations with NuRD complex components. (B) Chow-Ruskey Venn diagram of mTOR RIME datasets from four PCa cell lines in the basal state (vehicle-treated) shows a conserved set of 67 mTOR CIPs. (C) Basic functional annotation of the conserved mTOR 67-CIP hub identified in (B). (D) Metascape interactome network of the conserved mTOR 67-CIP set identified in (B) along with four extracted MCODE subcomplexes and their associated biological functions. Larger node size reflects increased protein interconnectivity. (E) Ranked normalized TPIs of identified mTOR RIME CIPs in four PCa cell lines from triplicate experiments of vehicle-treated versus immunoglobulin G control showing SUMO2 and SUMO3 as top conserved hits. See also Figures S3 and S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER ON-TARGETplus SMARTpool non-targeting control siRNAs Dharmacon (Horizon Discovery) Cat# D-001810-10-20 Human shHDAC2#1: CAGTCTCACCAATTT CAGAAA Sigma-Aldrich Cat # TRCN0000004819 Human shHDAC2#2: GCCTATTATCTCAAA GGTGAT Sigma-Aldrich Cat# TRCN0000004821 Human gene-specific primers for qRT-PCR analysis This paper Table S7 Human gene-specific primers for ChIP-qPCR analysis This paper Table S7 Recombinant DNA ViraPowerTM Lentiviral Packaging Mix Thermo Fisher Scientific Cat# K4975-00 Lentiviral packaging plasmid psPAX2 addgene Cat# 12260 VSV-G envelope expressing plasmid pMD2.G addgene Cat# 12259 mTOR shRNA plasmid addgene Cat# 1856 Software and algorithms Proteome Discoverer v2.3 Thermo Scientific Cat# OPTON-20141 Mascot v2.6 Matrix Science http://www.matrixscience.com/ Scaffold v4.10.0 Proteome Software https://www.proteomesoftware.com/ Phantasus v1.11.0 Bioconductor (https://doi.org/ 10.18129/B9.bioc.phantasus) https://genome.ifmo.ru/phantasus ExPASy PeptideCutter Duvaud et al., 2021 https://web.expasy.org/peptide_cutter/ WebGestalt Liao et al., 2019 RRID:SCR_006786 Ingenuity Pathway Analysis (IPA) QIAGEN RRID:SCR_008653 Metascape Zhou et al., 2019 RRID:SCR_016620 Intervene Khan and Mathelier, 2017 https://asntech.shinyapps.io/intervene/ ChIP-Atlas Oki et al., 2018 RRID:SCR_015511 HOMER v4.11 Heinz et al., 2010 RRID:SCR_010881 BioGRID v3.5 Oughtred et al., 2019 RRID:SCR_007393 STRING v11.0 Szklarczyk et al., 2019 RRID:SCR_005223 HuRI Luck et al., 2020 RRID:SCR_015670 IID Kotlyar et al., 2016 http://ophid.utoronto.ca/iid Morpheus Broad Institute RRID:SCR_017386 Cancer Dependency Map (DepMap) portal Broad Institute RRID:SCR_017655 Prism v9 GraphPad https://www.graphpad.com/ scientific-software/prism/ Other Easy-nLC II system Thermo Fisher Scientific (Proxeon Biosystems) https://www.thermofisher.com/ LTQ Orbitrap Velos spectrometer Thermo Fisher Scientific https://www.thermofisher.com/ ChemiDoc MP imaging system Bio-Rad Cat# 12003154 LightCycler 480 system Roche https://diagnostics.roche.com/ global/en/products/systems/lightcycler480-system.html

Techniques: Functional Assay, Control

Journal: Cell reports

Article Title: The mTOR chromatin-bound interactome in prostate cancer.

doi: 10.1016/j.celrep.2022.110534

Figure Lengend Snippet: Figure 7. Androgens promote assembly of an mTOR-AR-HDAC2 transcriptional complex (A) Co-IP experiments in LNCaP cells show that mTOR and AR interact with NuRD complex components. (B) R1881 increases the overlap of mTOR, AR, and HDAC2 ChIP-seq peaks in PCa cells. (C) Genome-wide mapping of mTOR-AR-HDAC2 co-occupied sites between vehicle (EtOH)- and R1881-treated PCa cells. (D) Pie charts showing that most of the mTOR-AR-HDAC2 peaks found within ±20 kb of ARG TSSs are up-regulated by androgens. (E) Genome browser views showing R1881-mediated de novo formation of an mTOR-AR-HDAC2 complex at ARGs in PCa cells. (F) Heatmaps of ChIP-qPCR enrichment values in LNCaP cells showing the temporal R1881-mediated co-recruitment of mTOR, AR, NuRD-associated HDAC2 and CHD4, RNA polymerase II, and deposition of the active histone mark H3K27ac at mTOR-AR-HDAC2-targeted ARGs shown in (E). (G) Immunoblots showing efficacy of shRNA-mediated silencing of HDAC2 in LNCaP cells. Lamin B1 levels are shown as a loading control.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER ON-TARGETplus SMARTpool non-targeting control siRNAs Dharmacon (Horizon Discovery) Cat# D-001810-10-20 Human shHDAC2#1: CAGTCTCACCAATTT CAGAAA Sigma-Aldrich Cat # TRCN0000004819 Human shHDAC2#2: GCCTATTATCTCAAA GGTGAT Sigma-Aldrich Cat# TRCN0000004821 Human gene-specific primers for qRT-PCR analysis This paper Table S7 Human gene-specific primers for ChIP-qPCR analysis This paper Table S7 Recombinant DNA ViraPowerTM Lentiviral Packaging Mix Thermo Fisher Scientific Cat# K4975-00 Lentiviral packaging plasmid psPAX2 addgene Cat# 12260 VSV-G envelope expressing plasmid pMD2.G addgene Cat# 12259 mTOR shRNA plasmid addgene Cat# 1856 Software and algorithms Proteome Discoverer v2.3 Thermo Scientific Cat# OPTON-20141 Mascot v2.6 Matrix Science http://www.matrixscience.com/ Scaffold v4.10.0 Proteome Software https://www.proteomesoftware.com/ Phantasus v1.11.0 Bioconductor (https://doi.org/ 10.18129/B9.bioc.phantasus) https://genome.ifmo.ru/phantasus ExPASy PeptideCutter Duvaud et al., 2021 https://web.expasy.org/peptide_cutter/ WebGestalt Liao et al., 2019 RRID:SCR_006786 Ingenuity Pathway Analysis (IPA) QIAGEN RRID:SCR_008653 Metascape Zhou et al., 2019 RRID:SCR_016620 Intervene Khan and Mathelier, 2017 https://asntech.shinyapps.io/intervene/ ChIP-Atlas Oki et al., 2018 RRID:SCR_015511 HOMER v4.11 Heinz et al., 2010 RRID:SCR_010881 BioGRID v3.5 Oughtred et al., 2019 RRID:SCR_007393 STRING v11.0 Szklarczyk et al., 2019 RRID:SCR_005223 HuRI Luck et al., 2020 RRID:SCR_015670 IID Kotlyar et al., 2016 http://ophid.utoronto.ca/iid Morpheus Broad Institute RRID:SCR_017386 Cancer Dependency Map (DepMap) portal Broad Institute RRID:SCR_017655 Prism v9 GraphPad https://www.graphpad.com/ scientific-software/prism/ Other Easy-nLC II system Thermo Fisher Scientific (Proxeon Biosystems) https://www.thermofisher.com/ LTQ Orbitrap Velos spectrometer Thermo Fisher Scientific https://www.thermofisher.com/ ChemiDoc MP imaging system Bio-Rad Cat# 12003154 LightCycler 480 system Roche https://diagnostics.roche.com/ global/en/products/systems/lightcycler480-system.html

Techniques: Co-Immunoprecipitation Assay, ChIP-sequencing, Genome Wide, ChIP-qPCR, Western Blot, shRNA, Control