Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: Decreasing caveolin 1 expression attenuates 17β-estradiol-mediated activation of eNOS. PAEC were transfected with either a caveolin 1 siRNA or a scrambled siRNA (as a control). Immunoblot analysis demonstrates a significant decrease in caveolin 1 protein levels in caveolin 1 siRNA-transfected cells (A and B; *, P < 0.05 vs. scrambled siRNA). Plasma membrane fractions were isolated and subjected to immunoblotting with either an antibody against ERα or eNOS. Decreasing caveolin 1 expression results in a significant reduction in ERα localization to the plasma membrane (C and D; *, P < 0.05 vs. scrambled siRNA) although total ERα levels are unchanged (C). The siRNA-mediated knockdown of caveolin 1 does not alter eNOS localized to the plasma membrane (E and F), Immunoblotting with the plasma membrane marker 5′-nucleotidase was also carried out to normalize for protein loading of the plasma membrane fractions (C and E). The siRNA-mediated knockdown of caveolin 1 significantly inhibits 17β-estradiol-stimulated (100 nm, 30 min) eNOS phosphorylation at serine 1177 (G and H) and NO generation (I). The siRNA-mediated knockdown of caveolin 1 does not inhibit the increase in NO generation in response to stimulation by shear stress (20 dyn/cm2, 15 min; panel J) or VEGF (100 ng/ml, 15min; panel K). Data are mean ± sem, n = 3–6. *, P < 0.05 vs. no 17β-estradiol; †, P < 0.05 vs. 17β-estradiol-treated scrambled siRNA.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Expressing, Activation Assay, Transfection, Western Blot, Isolation, Marker

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: 17β-Estradiol stimulates caveolin 1 phosphorylation through the activation of pp60Src. PAEC were exposed to pp60Src inhibitor, PP2 (10 μm, 30 min), before 17β-estradiol (100 nm, 30 min). 17β-Estradiol significantly increases phospho-Tyr14 caveolin 1, and PP2 inhibits this phosphorylation although total caveolin 1 levels are unchanged (A and B). 17β-Estradiol also produces significantly higher eNOS phosphorylation at serine 1177 compared with untreated cells, and pretreatment with the ER antagonist, ICI 182 780 (10 μm, 30 min) or PP2 significantly attenuates eNOS phosphorylation at serine 1177 (C and D). 17β-Estradiol treatment also induces a significant increase in NO generation, and pretreatment with ICI 182,780 or PP2 results in significant reduction in NO production (E). Data are mean ± sem n = 3–4. *, P < 0.05 vs. untreated; †, P < 0.05 vs. 17β-estradiol treated.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Activation Assay

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: Caveolin 1 phosphorylation is not required for the localization of ERα to the plasma membrane. Immunoblot analysis of PAEC transduced with adenoviral constructs for GFP (AdGFP), wild-type caveolin 1 (AdWTCav) or a tyrosine 14-deficient mutant (AdY14FCav) indicates significant overexpression of AdWTCav and AdY14Cav (A and B). There is significantly higher ERα in the plasma membrane in both AdWTCav- and AdY14Cav-transduced cells (C and D). AdY14Cav overexpression did not alter eNOS localization to the plasma membrane (E and F). Immunoblotting with the plasma membrane marker 5′-nucleotidase was also carried out to normalize for protein loading of the plasma membrane fractions (C and E). Immunofluorescence analysis of cells transduced with AdGFP, AdWTCav-1, or AdY14FCav-1 were then transfected with an ERα-GFP construct. The plasma membrane of these cells was labeled with Alexa Fluor 594 WGA (red). The extent of membrane localization of ERα was then determined by measuring the intensity of yellow fluorescence (overlap of red fluorescence of Alexa Fluor 594 WGA and green fluorescence of ERα-GFP (G); the white bar in each individual panel represents 30 μm). The intensity of yellow fluorescence is significantly higher in cells transduced with either AdWTCav or AdY14Cav (H). Data are mean ± sem n = 3. *, P < 0.05 vs. AdGFP-transduced cells.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Western Blot, Transduction, Construct, Mutagenesis, Over Expression, Marker, Immunofluorescence, Transfection, Labeling, Fluorescence

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: Caveolin 1 phosphorylation enhances the interaction of ERα with PI3 kinase. PAEC were transduced with AdGFP, AdWTCav-1, or AdY14FCav-1, exposed or not to 17β-estradiol, and the interaction of ERα with caveolin 1 (A and C) or PI3 kinase (A and B) was determined. 17β-Estradiol exposure causes a dissociation of the ERα-caveolin 1 complex (A and C). This dissociation was enhanced in cells overexpressing wild-type caveolin 1 and attenuated in cells overexpressing Y14F caveolin 1 (A and C). 17β-Estradiol also significantly increases the interaction of ERα with PI3 kinase (A and B). Transduction with AdWTCav potentiates the 17β-estradiol-mediated interaction (A and B) whereas AdY14FCav attenuates this effect (A and B). Data are mean ± sem; n = 3. *, P < 0.05 vs. untreated cells; †, P < 0.05 vs. AdGFP with 17β-estradiol. IB, Immunoblotting; IP, immunoprecipitation.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Transduction, Western Blot, Immunoprecipitation

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: Caveolin 1 phosphorylation enhances Akt activation and NO signaling in response to 17β-estradiol. PAEC were transduced with AdGFP, AdWTCav-1, or AdY14FCav-1, exposed or not to 17β-estradiol, and the effect on Akt activity (A and B), eNOS phosphorylation at serine1177 (C and D), and NO generation (E) was determined. The 17β-estradiol-mediated increase in Akt activity (A and B), eNOS phosphorylation at serine 1177 (C and D), and NO generation (E) is enhanced in cells overexpressing wild-type caveolin 1 and attenuated in cells overexpressing Y14F caveolin 1. The overexpression of wild-type caveolin 1 (F and G) or AdY14F (H and I) did not significantly attenuate the increase in NO generation in response to stimulation by shear stress (20 dyn/cm2, 15 min) or VEGF (100 ng/ml, 15min). Data are mean ± sem; n =3–6. *, P < 0.05 vs. untreated; †, P < 0.05 vs. AdGFP with 17β-estradiol.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Activation Assay, Transduction, Activity Assay, Over Expression

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: The scaffold domain of caveolin 1 is required for ERα trafficking to the plasma membrane but not for protein-protein interaction. Immunoblot analysis of PAEC transduced with adenoviral constructs for GFP (AdGFP), wild-type caveolin 1 (AdWTCav), or a caveolin 1 mutant lacking the scaffold domain (AdCavΔ) indicates significant overexpression of AdWTCav and AdCavΔ (A and B). There is significantly higher ERα (C and D) and caveolin 1 (C and E) in the plasma membrane in the AdWTCav-transduced cells. However, both ERα (C and D) and caveolin 1 (C and E) are significantly decreased in the AdCavΔ-transduced cells (C and E). Immunoblotting (IB) with the plasma membrane marker 5′-nucleotidase was also carried out to normalize for protein loading of the plasma membrane fractions (C). Immunoprecipitation (IP) of caveolin 1 from whole-cell extracts followed by IB with ERα identified a significant increase in ERα bound to caveolin 1 in both AdWTCav- and AdCavΔ-transduced cells (F and G). Data are mean ± sem; n = 3. *, P < 0.05 vs. AdGFP-transduced cells.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Western Blot, Transduction, Construct, Mutagenesis, Over Expression, Marker, Immunoprecipitation

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: Amino acids 62–73 of caveolin 1 span the ERα interaction region. Probing a peptide array representing the entire sequence of human caveolin 1 with recombinant human ERα (100 ng/ml) identified a single highly reactive peptide spanning amino acids 62–73 (shown by arrow in panel A). TAT-Cav62–73, along with a scrambled version containing a similar amino acid composition (TAT-Cav62–73scr), was synthesized as a fusion with the N-terminal TAT protein transduction domain from the HIV (B). PAEC were exposed to each peptide (100 ng/ml). Immunopreciptation (IP) analyses demonstrate that TAT-Cav62–73 significantly attenuates the association of ERα with caveolin 1 (C and D). Western blot analysis also demonstrates that TAT-Cav62–73 significantly attenuates ERα levels in the plasma membrane without altering total ERα levels (E and F; *, P < 0.05 vs. TAT-Cav62–73scr). Immunoblotting (IB) with the plasma membrane marker 5′-nucleotidase was also carried out to normalize for protein loading of the plasma membrane fractions (E). TAT-Cav62–73 and TAT-Cav62–73scr peptide-treated cells were transfected with an ERα-GFP construct and the plasma membrane labeled with Alexa Fluor 594 WGA (red). The extent of plasma membrane localization of ERα was determined by measuring the intensity of yellow fluorescence (overlap of red fluorescence of Alexa Fluor 594 WGA, and green fluorescence of ERα-GFP). TAT-Cav62–73 significantly attenuates the yellow fluorescence (G and H, the white bar in each panel represents 30 μm). TAT-Cav62–73 did not alter eNOS localization to the plasma membrane (I and J). n = 3; *, P < 0.05 vs. TAT-Cav62–73scr.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Peptide Microarray, Sequencing, Recombinant, Synthesized, Transduction, Western Blot, Marker, Transfection, Construct, Labeling, Fluorescence

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: Disrupting ERα-caveolin 1 interactions attenuates the interaction of ERα with PI3 kinase and inhibits downstream NO signaling. TAT-Cav62–73 significantly attenuates the 17β-estradiol-mediated association of ERα with PI3 kinase (A and B), the increase in eNOS phosphorylation at serine 1177 (C and D), and the increase in NO generation (E). TAT-Cav62–73 did inhibit the increase in NO generation in response to stimulation by shear stress (20 dyn/cm2, 15 min; panel F) or VEGF (100 ng/ml, 15min; panel G). Data are mean ± sem; n = 3–6. *, P < 0.05 vs. untreated; †, P < 0.05 vs. TAT-Cav62–73scr with 17β-estradiol.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques:

Journal: bioRxiv

Article Title: Antigen-derived peptides directly engage the unfolded-protein sensor IRE1α to curb cross-presentation by dendritic cells

doi: 10.1101/2021.09.10.459738

Figure Lengend Snippet: (A) Polystyrene wells were coated with native or heat-denatured ovalbumin (10 μg/ml) and incubated with purified recombinant human IRE1α LD-Fc fusion protein (10 μg/ml), followed by colorimetric detection with an HRP-conjugated anti-human Fc antibody. (B) Native or heat-denatured ovalbumin at indicated concentrations was incubated with IRE1α LD-Fc (10 μg/ml), immunoprecipitated via monoclonal anti-IRE1α LD antibody, and analyzed by IB. (C) A tiled 18 aa-long peptide array spanning ovalbumin was incubated with IRE1α LD-Fc (500 nM) followed by colorimetric detection with an HRP-conjugated anti-human Fc antibody. (D) Biotin-tagged ovalbumin-based peptides (100 μM) were incubated with FLAG-tagged IRE1α LD (50 μM), cross-linked with disuccinimidyl suberate (DSS) and analyzed by IB. B1, B2, B3 are WT peptides; B1’, B2’ are mutant peptides in which all hydrophobic residues were replaced by aspartic acid. (E) HEK293 cells were transfected with cDNA constructs encoding Myc-tagged peptides (M1, M2, M3) derived from corresponding ovalbumin regions (B1, B2, B3) and containing an ER-directed signal sequence for 48 hr, followed by immunoprecipitation with anti-Myc antibody and IB for IRE1α or Myc. Bar graph indicates signal ratio for IRE1α over Myc. (F) U2OS cells were transfected with cDNA constructs encoding Myc-tagged peptides (M1.1, M1.2, M1.3, M2.1, M2.2, M2.3, M3) derived from corresponding ovalbumin regions (B1, B2, B3) and containing an ER-targeting signal sequence for 48 hr, followed by real-time quantitative PCR (RT-qPCR) analysis for XBP1s, XBP1u, CD59, and DGAT2 mRNA levels. Bar graphs in panels A , C and F represent mean ± SD from three independent technical repeats; images in panels B, D and E represent at least two similar experiments.

Article Snippet: Antibodies used for western blot analysis include IRE1α (3294, Cell Signaling Technology, Danvers, MA), β-actin (5125, Cell Signaling), ATF6 (66563-1-Ig, Proteintech, Rosemont, IL), TAP1 (12341, Cell Signaling) CHOP (2895, Cell Signaling), ATF4 (11815, Cell Signaling), ovalbumin (P1-196, Thermo-Fisher), Myc tag (2272, Cell Signaling) and biotin (5597, Cell Signaling).

Techniques: Incubation, Purification, Recombinant, Immunoprecipitation, Peptide Microarray, Mutagenesis, Transfection, Construct, Derivative Assay, Sequencing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Antigen-derived peptides directly engage the unfolded-protein sensor IRE1α to curb cross-presentation by dendritic cells

doi: 10.1101/2021.09.10.459738

Figure Lengend Snippet: (A) A tiled 18 aa-long peptide array spanning GP70 was incubated with IRE1α LD-Fc (500 nM) followed by colorimetric detection with an HRP-conjugated anti-human Fc antibody. (B) Sequences of biotin-tagged peptides (labeled B) used in ; Myc-tagged signal peptides (labeled M) used in ; and Myc-tagged peptides (Labeled M) used in . Bar graphs in panel A represents mean ± SD from three independent technical repeats.

Article Snippet: Antibodies used for western blot analysis include IRE1α (3294, Cell Signaling Technology, Danvers, MA), β-actin (5125, Cell Signaling), ATF6 (66563-1-Ig, Proteintech, Rosemont, IL), TAP1 (12341, Cell Signaling) CHOP (2895, Cell Signaling), ATF4 (11815, Cell Signaling), ovalbumin (P1-196, Thermo-Fisher), Myc tag (2272, Cell Signaling) and biotin (5597, Cell Signaling).

Techniques: Peptide Microarray, Incubation, Labeling

Journal: bioRxiv

Article Title: Antigen-derived peptides directly engage the unfolded-protein sensor IRE1α to curb cross-presentation by dendritic cells

doi: 10.1101/2021.09.10.459738

Figure Lengend Snippet: (A) Consensus stem-loop endomotifs for RIDD recognition found in murine MHC-I heavy-chain H-2K, H-2D and H-2L mRNA sequences. (B) Purified recombinant IRE1α kinase-endoribonucleae (KR) protein was incubated with RNA transcripts of H-2K, H-2D and H-2L, in absence or presence of G9668 (10 μM), followed by agarose gel electrophoresis to determine transcript integrity. (C, D) BMDCs were treated with actinomycin D (2 μg/ml) to block de novo transcription and pulsed with ovalbumin (500 μg/ml) combined with DMSO, G9668 (3 μM), or 4μ8C (1 μM) for the indicated time period (C) or for 8 hr (D) , followed by RT-qPCR analysis of the indicated transcripts. Analysis was performed using unpaired, two-tailed t test, * P ≤ 0.05. Panel B image represents three similar experiments; bar graphs in panels C and D represent mean ± SD from three independent biological repeats.

Article Snippet: Antibodies used for western blot analysis include IRE1α (3294, Cell Signaling Technology, Danvers, MA), β-actin (5125, Cell Signaling), ATF6 (66563-1-Ig, Proteintech, Rosemont, IL), TAP1 (12341, Cell Signaling) CHOP (2895, Cell Signaling), ATF4 (11815, Cell Signaling), ovalbumin (P1-196, Thermo-Fisher), Myc tag (2272, Cell Signaling) and biotin (5597, Cell Signaling).

Techniques: Purification, Recombinant, Incubation, Agarose Gel Electrophoresis, Blocking Assay, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Antigen-derived peptides directly engage the unfolded-protein sensor IRE1α to curb cross-presentation by dendritic cells

doi: 10.1101/2021.09.10.459738

Figure Lengend Snippet: (A, B) Animals were inoculated s.c. with parental or IRE1α KO CT26 cells and tumor growth was measured over 27 days. (A) Final day tumor measurements, Scatter plots represent mean ± SD. (B) IB analysis of IRE1α expression and activation. (C, D) Mice were inoculated s.c. with CT26 cells, grouped out 7 days afterwards and treated with vehicle or G9668 (250 mg/kg, BID). (C) Growth trajectories of CT26 tumors in individual vehicle- and G9668-treated animals over 17 days and IB analysis of total tumor lysates (D) are depicted. Number of animals included in each study is noted in corresponding tables.

Article Snippet: Antibodies used for western blot analysis include IRE1α (3294, Cell Signaling Technology, Danvers, MA), β-actin (5125, Cell Signaling), ATF6 (66563-1-Ig, Proteintech, Rosemont, IL), TAP1 (12341, Cell Signaling) CHOP (2895, Cell Signaling), ATF4 (11815, Cell Signaling), ovalbumin (P1-196, Thermo-Fisher), Myc tag (2272, Cell Signaling) and biotin (5597, Cell Signaling).

Techniques: Expressing, Activation Assay

Journal: bioRxiv

Article Title: Antigen-derived peptides directly engage the unfolded-protein sensor IRE1α to curb cross-presentation by dendritic cells

doi: 10.1101/2021.09.10.459738

Figure Lengend Snippet: (A-B) Animals were inoculated s.c. with parental or IRE1α KO 4T1 cells and tumor growth was monitored over 25 days, with final measurements (A) and IB analysis of total tumor lysates (B) presented. (C-E) Mice were inoculated s.c. with 4T1 cells, grouped out 7 days afterwards and treated with vehicle or G9668 (250 mg/kg, BID). (C) Tumor growth in individual animals was measured over 19 days, and (D) IRE1α expression and activation were analyzed by IB. (E, F) Mice were treated with vehicle or G9668 for 6 days and tumors were then analyzed. (E) Transcript levels of indicated genes characterized as RIDD targets in tumor-infiltrating DCs. (F) Relative abundance of group 1 (Hcar) and group 2 (Hilpda) tumor-infiltrating monocytes (Mc.) and M1- or M2-polarized macrophages in vehicle- and G9668- treated animals, analyzed by single-cell RNA sequencing data. *P ≤ 0.05, **P ≤ 0.01 ***P ≤ 0.001, ****P ≤ 0.0001. Number of animals included in each study is noted in corresponding tables. Scatter plots in panels A , E and F represent mean ± SD.

Article Snippet: Antibodies used for western blot analysis include IRE1α (3294, Cell Signaling Technology, Danvers, MA), β-actin (5125, Cell Signaling), ATF6 (66563-1-Ig, Proteintech, Rosemont, IL), TAP1 (12341, Cell Signaling) CHOP (2895, Cell Signaling), ATF4 (11815, Cell Signaling), ovalbumin (P1-196, Thermo-Fisher), Myc tag (2272, Cell Signaling) and biotin (5597, Cell Signaling).

Techniques: Expressing, Activation Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Antigen-derived peptides directly engage the unfolded-protein sensor IRE1α to curb cross-presentation by dendritic cells

doi: 10.1101/2021.09.10.459738

Figure Lengend Snippet: (A-E) Mice were inoculated s.c. with 4T1 cells, grouped out 7 days afterwards and treated with vehicle or G9668 (250 mg/kg, BID). (A) Tumor growth trajectories were measured over 19 days (n = 15). (B-E) Mice were treated with G9668 for 6 days and tumor-infiltrating leukocytes were then analyzed. (B) Representation of tumor-infiltrating leukocytes by t-distributed stochastic neighbor embedding (t-SNE) plot from single-cell RNA sequencing data. (C) Abundance of tumor-infiltrating DCs and CD8 + T cells in vehicle- and G9668-treated animals. (D) Transcript levels of indicated genes in tumor-infiltrating DCs. (E) Mice were inoculated s.c. with parental or IRE1α KO 4T1 cells, grouped out 7 days afterwards and treated with vehicle or G9668 (250 mg/kg) (n = 5). Abundance of CD8 + T cells in the tumor and expression of indicated activation markers were assayed by flow cytometry. Analysis was performed using one-way ANOVA for panel A and unpaired, two-tailed t test for panels C - E , *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. Scatter plots represent mean ± SD.

Article Snippet: Antibodies used for western blot analysis include IRE1α (3294, Cell Signaling Technology, Danvers, MA), β-actin (5125, Cell Signaling), ATF6 (66563-1-Ig, Proteintech, Rosemont, IL), TAP1 (12341, Cell Signaling) CHOP (2895, Cell Signaling), ATF4 (11815, Cell Signaling), ovalbumin (P1-196, Thermo-Fisher), Myc tag (2272, Cell Signaling) and biotin (5597, Cell Signaling).

Techniques: RNA Sequencing, Expressing, Activation Assay, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Antigen-derived peptides directly engage the unfolded-protein sensor IRE1α to curb cross-presentation by dendritic cells

doi: 10.1101/2021.09.10.459738

Figure Lengend Snippet: (A, B) Mice were inoculated orthotopically with WT or IRE1α KO EMT6 cells and tumor growth was measured over 24 days, with final tumor measurements (scatter plots represent mean ± SD) (A) and IB analysis of total tumor lysates (B) presented. (C, D) Mice were inoculated with WT or IRE1α KO EMT6 cells, grouped out 7 days afterwards and treated with vehicle, G9668 (250 mg/kg, BID), anti-PD-L1 antibody (10 mg/kg at first dose, 5 mg/kg BIW thereafter), or the combination. (C) Tumor growth trajectories were measured over 19 days. ( D) IRE1α activation was analyzed by IB. Number of animals included in each study is noted in corresponding tables.

Article Snippet: Antibodies used for western blot analysis include IRE1α (3294, Cell Signaling Technology, Danvers, MA), β-actin (5125, Cell Signaling), ATF6 (66563-1-Ig, Proteintech, Rosemont, IL), TAP1 (12341, Cell Signaling) CHOP (2895, Cell Signaling), ATF4 (11815, Cell Signaling), ovalbumin (P1-196, Thermo-Fisher), Myc tag (2272, Cell Signaling) and biotin (5597, Cell Signaling).

Techniques: Activation Assay

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-HOXA9 , Novus Biologicals , Cat# NBP2-32356.

Techniques: Recombinant, Blocking Assay, Lysis, SYBR Green Assay, Reporter Assay, Extraction, Bicinchoninic Acid Protein Assay, cDNA Synthesis, Reverse Transcription, TaqMan microRNA Assay, Mass Spectrometry, Microarray, Gene Expression, Retroviral, Negative Control, Plasmid Preparation, Software, Multiplex sample analysis