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Addgene inc pegfp n1 rack1 plasmid

(A) Relative Expression of IL-8 in Caco2-BBE cells with and without Nox1 inhibitor in response to IL-1β. (B) Relative Expression of IL-8 in Caco2-BBE cells with and without Nox1 inhibitor in response to damage and pathogen associated molecules. (C) Relative Expression of IL-8 in NOX1 -/- and WT mouse colonoids in response to IL-1β. (D) Schematic showing the experimental outline for generation of colonoids and the redox-proteomics (E) Scatter plot showing the results of the proteomics screen with differentially oxidized cysteine residues of proteins in NOX1 -/- and WT organoids in the X and Y axis. The significantly altered candidates are highlighted in blue. (F) Scatter plot showing the redox-modified cysteine residues from the proteomics screen. Cysteines from GNB2L1 <t>(RACK1)</t> are highlighted in oranges. (G) Ribbon and space filling model of crystal structure of RACK1 showing the relative positions of the different cysteines. (H) H/L ratios of the individual cysteines of RACK1 in both WT and NOX1 -/- . (I) Conservation plot of RACK1 from lower eukaryotes to human. Values represent the conservation score from multiple sequence alignment. Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t tests in (A) and (B); ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001

Pegfp N1 Rack1 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pegfp n1 rack1 plasmid - by Bioz Stars, 2026-05

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1) Product Images from "Trans-kingdom coupling of redox signaling to environmental cell stress responses through multiphase partitioning"

Article Title: Trans-kingdom coupling of redox signaling to environmental cell stress responses through multiphase partitioning

Journal: bioRxiv

doi: 10.1101/2025.05.09.653213

Figure Legend Snippet: (A) Relative Expression of IL-8 in Caco2-BBE cells with and without Nox1 inhibitor in response to IL-1β. (B) Relative Expression of IL-8 in Caco2-BBE cells with and without Nox1 inhibitor in response to damage and pathogen associated molecules. (C) Relative Expression of IL-8 in NOX1 -/- and WT mouse colonoids in response to IL-1β. (D) Schematic showing the experimental outline for generation of colonoids and the redox-proteomics (E) Scatter plot showing the results of the proteomics screen with differentially oxidized cysteine residues of proteins in NOX1 -/- and WT organoids in the X and Y axis. The significantly altered candidates are highlighted in blue. (F) Scatter plot showing the redox-modified cysteine residues from the proteomics screen. Cysteines from GNB2L1 (RACK1) are highlighted in oranges. (G) Ribbon and space filling model of crystal structure of RACK1 showing the relative positions of the different cysteines. (H) H/L ratios of the individual cysteines of RACK1 in both WT and NOX1 -/- . (I) Conservation plot of RACK1 from lower eukaryotes to human. Values represent the conservation score from multiple sequence alignment. Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t tests in (A) and (B); ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001

Techniques Used: Expressing, Modification, Sequencing, Two Tailed Test

(A) Biotin switch immunoprecipitation and immunoblot with anti-RACK1 showing increased proportion of oxidized RACK1 following IL-1β (10 ng/μL) in WT versus NOX1 knockout colonic epithelial cells. (B) NF-κB activity in HEK cells expressing scrambled vector (EV), or knockdown of RACK1 (siRNA) following IL-1β (10 ng/μL). ***p < 0.001

Figure Legend Snippet: (A) Biotin switch immunoprecipitation and immunoblot with anti-RACK1 showing increased proportion of oxidized RACK1 following IL-1β (10 ng/μL) in WT versus NOX1 knockout colonic epithelial cells. (B) NF-κB activity in HEK cells expressing scrambled vector (EV), or knockdown of RACK1 (siRNA) following IL-1β (10 ng/μL). ***p < 0.001

Techniques Used: Immunoprecipitation, Western Blot, Knock-Out, Activity Assay, Expressing, Plasmid Preparation, Knockdown

(A) NF-κB activity in HEK cells stimulated with IL-1β (10 ng/μL) or with control stimulation with pretreatment with either media, NAC (5mM) or 1mM Rotenone (1mM). (B) NF-κB activity in HEK cells transfected with RACK1-WT, one of the cysteine mutants or empty vector (ev) stimulated with IL-1β (10 ng/ml) or with control. (C) NF-κB activity in HEK transfected with RACK1-WT, C286A mutant or empty vector (ev)with varying dose of IL-1β. (D) p65 immunofluorescence staining in empty vector, RACK1-WT or RACK1-C286A transfected HEK cells either with mock or IL-1β (10 ng/ml) stimulation for 30 minutes. Nuclei was stained using Hoescht stain. (F) Quantification of immunofluorescence image from (D). (G) Western blot graph showing relative p65 phosphorylation in HEK293T expressing either empty vector (ev) (H) Time series of co-immunoprecipitation of IKKα with overexpressed wtRACK1 in HEK293T following IL-1β (10 ng/μL) stimulation. (I) Co-immunoprecipitation of IKKα with RACK1 in HEK293T expressing empty vector (ev), wt or cysteine mutants RACK1 following IL-1β (10 ng/μL,1h) stimulation. (J) NF-κB activity in HEK cells expressing empty vector (ev), wt or cysteine mutants RACK1 stimulated with TNFα, LPS or unstimulated. *p < 0.05, **p < 0.01, ***p < 0.001

Figure Legend Snippet: (A) NF-κB activity in HEK cells stimulated with IL-1β (10 ng/μL) or with control stimulation with pretreatment with either media, NAC (5mM) or 1mM Rotenone (1mM). (B) NF-κB activity in HEK cells transfected with RACK1-WT, one of the cysteine mutants or empty vector (ev) stimulated with IL-1β (10 ng/ml) or with control. (C) NF-κB activity in HEK transfected with RACK1-WT, C286A mutant or empty vector (ev)with varying dose of IL-1β. (D) p65 immunofluorescence staining in empty vector, RACK1-WT or RACK1-C286A transfected HEK cells either with mock or IL-1β (10 ng/ml) stimulation for 30 minutes. Nuclei was stained using Hoescht stain. (F) Quantification of immunofluorescence image from (D). (G) Western blot graph showing relative p65 phosphorylation in HEK293T expressing either empty vector (ev) (H) Time series of co-immunoprecipitation of IKKα with overexpressed wtRACK1 in HEK293T following IL-1β (10 ng/μL) stimulation. (I) Co-immunoprecipitation of IKKα with RACK1 in HEK293T expressing empty vector (ev), wt or cysteine mutants RACK1 following IL-1β (10 ng/μL,1h) stimulation. (J) NF-κB activity in HEK cells expressing empty vector (ev), wt or cysteine mutants RACK1 stimulated with TNFα, LPS or unstimulated. *p < 0.05, **p < 0.01, ***p < 0.001

Techniques Used: Activity Assay, Control, Transfection, Plasmid Preparation, Mutagenesis, Immunofluorescence, Staining, Western Blot, Phospho-proteomics, Expressing, Immunoprecipitation

(A) Representative fluorescence image showing RACK1 condensates in HCT-8 cells expressing RACK1-GFP and stimulated with IL-1β (10 ng/μL) or with 17-AAG (10 µM) but not in untreated cells. (B) Representative fluorescence image showing time course of RACK1 condensate formation in HCT-8 cells expressing RACK1-GFP stimulated with IL-1β (10 ng/μL). (C) Fluorescence recovery after after photobleaching (FRAP) time course of RACK1-GFP condensates after IL-1β (10 ng/μL) stimulation(orange) and unstimulated cytosolic RACK1-GFP(black). The solid line represents the mean and the dash line represents the error bounds defined by the standard deviation. (D) Representative fluorescence image showing colocalization of RACK1-GFP and INAVA-mcherry in HCT-8 cells upon stimulation with IL-1β (10 ng/μL) or with 17-AAG (10 µM). (E) Pearson correlation of coefficient to quantify colocalization between RACK1-GFP and INAVA-mcherry in HCT-8 cells upon stimulation with IL-1β (10 ng/μL) or with 17-AAG (10 µM). (F) Representative Co-immunoprecipitation blots showing interaction between RACK1 and GFP at two different timepoints in unstimulated or when stimulated with IL-1β (10 ng/μL) or with 17-AAG (10 µM) (G) Quantification of (F). (H) Representative fluorescence image of Caco-2 cells, untreated or stimulated with IL-1β (10 ng/μL) and/or Nox1 inhibitor for 1 hour and stained with DCP-Rho1(10 μM). (K) Representative ratiometric fluorescence image of HCT-8 cells overexpressing RACK1-mcherry. The intensity is the ratio of fluorescence signals excited by 488 nm and 405 nm lasers. (L) Quantification of intensity ratio described in (K) as a function of time. *p < 0.05, **p < 0.01, ***p < 0.001

Figure Legend Snippet: (A) Representative fluorescence image showing RACK1 condensates in HCT-8 cells expressing RACK1-GFP and stimulated with IL-1β (10 ng/μL) or with 17-AAG (10 µM) but not in untreated cells. (B) Representative fluorescence image showing time course of RACK1 condensate formation in HCT-8 cells expressing RACK1-GFP stimulated with IL-1β (10 ng/μL). (C) Fluorescence recovery after after photobleaching (FRAP) time course of RACK1-GFP condensates after IL-1β (10 ng/μL) stimulation(orange) and unstimulated cytosolic RACK1-GFP(black). The solid line represents the mean and the dash line represents the error bounds defined by the standard deviation. (D) Representative fluorescence image showing colocalization of RACK1-GFP and INAVA-mcherry in HCT-8 cells upon stimulation with IL-1β (10 ng/μL) or with 17-AAG (10 µM). (E) Pearson correlation of coefficient to quantify colocalization between RACK1-GFP and INAVA-mcherry in HCT-8 cells upon stimulation with IL-1β (10 ng/μL) or with 17-AAG (10 µM). (F) Representative Co-immunoprecipitation blots showing interaction between RACK1 and GFP at two different timepoints in unstimulated or when stimulated with IL-1β (10 ng/μL) or with 17-AAG (10 µM) (G) Quantification of (F). (H) Representative fluorescence image of Caco-2 cells, untreated or stimulated with IL-1β (10 ng/μL) and/or Nox1 inhibitor for 1 hour and stained with DCP-Rho1(10 μM). (K) Representative ratiometric fluorescence image of HCT-8 cells overexpressing RACK1-mcherry. The intensity is the ratio of fluorescence signals excited by 488 nm and 405 nm lasers. (L) Quantification of intensity ratio described in (K) as a function of time. *p < 0.05, **p < 0.01, ***p < 0.001

Techniques Used: Fluorescence, Expressing, Standard Deviation, Immunoprecipitation, Staining

(A) PDB structure of RACK1 almost no unstructured regions (B) PDB structure of INAVA as predicted by Alphafold showing large regions of highly unstructured regions and low -residue model confidence score. (C) Predicted Disorder score of RACK1 with and without changes in redox state of amino acids in protein.( , ) (D) Predicted Disorder score of INAVA with and without changes in redox state of amino acids in protein. Representative immunofluorescence image of HCT-8 overexpressing wt RACK1-mcherry and stained with G3BP and stimulated with 17AAG (10 µM) (E) and (F) IL-1β (10 ng/μL). (G) Pearson correlation coefficient showing colocalization of G3BP and Rack1. (E) shows a representative example with a cell with high colocalization and (F) represents a cell with low colocalization.

Figure Legend Snippet: (A) PDB structure of RACK1 almost no unstructured regions (B) PDB structure of INAVA as predicted by Alphafold showing large regions of highly unstructured regions and low -residue model confidence score. (C) Predicted Disorder score of RACK1 with and without changes in redox state of amino acids in protein.( , ) (D) Predicted Disorder score of INAVA with and without changes in redox state of amino acids in protein. Representative immunofluorescence image of HCT-8 overexpressing wt RACK1-mcherry and stained with G3BP and stimulated with 17AAG (10 µM) (E) and (F) IL-1β (10 ng/μL). (G) Pearson correlation coefficient showing colocalization of G3BP and Rack1. (E) shows a representative example with a cell with high colocalization and (F) represents a cell with low colocalization.

Techniques Used: Residue, Immunofluorescence, Staining

(A) Evolutionary distance of different eukaryotic RACK1 (B) Sequence alignment of 7WD domains of human RACK1. (C) RACK1 homologs across diverse kingdoms as a sunburst plot. (D) Sequence alignment of different bacterial homologs (E) WD7 sequence alignment between human and Entamoeba histolytica RACK1 showing conserved cysteine.

Figure Legend Snippet: (A) Evolutionary distance of different eukaryotic RACK1 (B) Sequence alignment of 7WD domains of human RACK1. (C) RACK1 homologs across diverse kingdoms as a sunburst plot. (D) Sequence alignment of different bacterial homologs (E) WD7 sequence alignment between human and Entamoeba histolytica RACK1 showing conserved cysteine.

Techniques Used: Sequencing

(A) Multiple Sequence alignment of amino acids proximal to cysteine 286 in eukaryotes. (B) Representative immunofluorescence image of fission yeast cells expressing Flag-cpc2 and treated with either 10% ethanol or media control for 10 minutes. (C) Quantification of number of puncta per cell in control and ethanol treated yeast. (D) Multiple sequence alignment of the amino acids proximal to cysteine 286 in human RACK1 against bacterial homologs. (E) Representative ratiometric fluorescence image of E. coli expressing bacterial analog of RACK1 tagged to Hyper3. (F) Quantification of intensity ratio of E. coli expressing bacterial analog of RACK1 tagged to Hyper3 when treated with either 10% ethanol or media control. (G) Representative fluorescence image of E. coli expressing human RACK1 protein tagged with GFP treated with either 10% ethanol or media control for 10 minutes. (H) Quantification of normalized coefficient of variance in GFP signal as a measure of heterogeneity of RACK1 signal in the cytosol. *p < 0.05, **p < 0.01, ***p < 0.001

Figure Legend Snippet: (A) Multiple Sequence alignment of amino acids proximal to cysteine 286 in eukaryotes. (B) Representative immunofluorescence image of fission yeast cells expressing Flag-cpc2 and treated with either 10% ethanol or media control for 10 minutes. (C) Quantification of number of puncta per cell in control and ethanol treated yeast. (D) Multiple sequence alignment of the amino acids proximal to cysteine 286 in human RACK1 against bacterial homologs. (E) Representative ratiometric fluorescence image of E. coli expressing bacterial analog of RACK1 tagged to Hyper3. (F) Quantification of intensity ratio of E. coli expressing bacterial analog of RACK1 tagged to Hyper3 when treated with either 10% ethanol or media control. (G) Representative fluorescence image of E. coli expressing human RACK1 protein tagged with GFP treated with either 10% ethanol or media control for 10 minutes. (H) Quantification of normalized coefficient of variance in GFP signal as a measure of heterogeneity of RACK1 signal in the cytosol. *p < 0.05, **p < 0.01, ***p < 0.001

Techniques Used: Sequencing, Immunofluorescence, Expressing, Control, Fluorescence

(A) String network analysis of redox-proteomics screen colored by manually selected subset of significant functional module. (B) Representative flow cytometry data showing intensity of puromycin incorporation in HCT-8 cells expressing either Rack-1 WT mCherry (top) or Rack-1 Call mCherry (bottom) treated with 17-AAG (10 µM, 10 minutes, orange curve) or untreated (blue). (C) Puromycin incorporation in HCT-8 cells expressing either Rack-1 WT mCherry or Rack-1 Call mCherry treated with 17-AAG (10 µM, 10 minutes) normalized to untreated. (D) NF-κB activity in HEK cells overexpressing empty vector, RACK1-WT or RACK1-C all stimulated with LPS (10 mg/ml). (E) Representative fluorescence image showing IRF3 translocation in HEK cells overexpressing empty vector, RACK1-WT or RACK1-Call stimulated with poly I:C (10µg/ml) (F) IP10 production following infection of HEK293T cells overexpressing empty vector, RACK1-WT, RACK1-C286A or RACK1-Call mutant with SeV (MOI = 1, 1h) (G) Representative ratiometric fluorescence image of Human duodenal organoids transfected with Rack1-Hyper3 and exposed to E. coli strains—pathogenic EPEC O127:H6, non-pathogenic, or uninfected. Quantification of number of puncta formed (H) and Mean Puncta Intensity (I) per transfected organoid. *p < 0.05, **p < 0.01, ***p < 0.001

Figure Legend Snippet: (A) String network analysis of redox-proteomics screen colored by manually selected subset of significant functional module. (B) Representative flow cytometry data showing intensity of puromycin incorporation in HCT-8 cells expressing either Rack-1 WT mCherry (top) or Rack-1 Call mCherry (bottom) treated with 17-AAG (10 µM, 10 minutes, orange curve) or untreated (blue). (C) Puromycin incorporation in HCT-8 cells expressing either Rack-1 WT mCherry or Rack-1 Call mCherry treated with 17-AAG (10 µM, 10 minutes) normalized to untreated. (D) NF-κB activity in HEK cells overexpressing empty vector, RACK1-WT or RACK1-C all stimulated with LPS (10 mg/ml). (E) Representative fluorescence image showing IRF3 translocation in HEK cells overexpressing empty vector, RACK1-WT or RACK1-Call stimulated with poly I:C (10µg/ml) (F) IP10 production following infection of HEK293T cells overexpressing empty vector, RACK1-WT, RACK1-C286A or RACK1-Call mutant with SeV (MOI = 1, 1h) (G) Representative ratiometric fluorescence image of Human duodenal organoids transfected with Rack1-Hyper3 and exposed to E. coli strains—pathogenic EPEC O127:H6, non-pathogenic, or uninfected. Quantification of number of puncta formed (H) and Mean Puncta Intensity (I) per transfected organoid. *p < 0.05, **p < 0.01, ***p < 0.001

Techniques Used: Functional Assay, Flow Cytometry, Expressing, Activity Assay, Plasmid Preparation, Fluorescence, Translocation Assay, Infection, Mutagenesis, Transfection

Image Search Results

Journal: bioRxiv

Article Title: Trans-kingdom coupling of redox signaling to environmental cell stress responses through multiphase partitioning

doi: 10.1101/2025.05.09.653213

Figure Lengend Snippet: (A) Relative Expression of IL-8 in Caco2-BBE cells with and without Nox1 inhibitor in response to IL-1β. (B) Relative Expression of IL-8 in Caco2-BBE cells with and without Nox1 inhibitor in response to damage and pathogen associated molecules. (C) Relative Expression of IL-8 in NOX1 -/- and WT mouse colonoids in response to IL-1β. (D) Schematic showing the experimental outline for generation of colonoids and the redox-proteomics (E) Scatter plot showing the results of the proteomics screen with differentially oxidized cysteine residues of proteins in NOX1 -/- and WT organoids in the X and Y axis. The significantly altered candidates are highlighted in blue. (F) Scatter plot showing the redox-modified cysteine residues from the proteomics screen. Cysteines from GNB2L1 (RACK1) are highlighted in oranges. (G) Ribbon and space filling model of crystal structure of RACK1 showing the relative positions of the different cysteines. (H) H/L ratios of the individual cysteines of RACK1 in both WT and NOX1 -/- . (I) Conservation plot of RACK1 from lower eukaryotes to human. Values represent the conservation score from multiple sequence alignment. Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t tests in (A) and (B); ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The pEGFP-N1-Rack1 plasmid was obtained from Addgene (#41088).

Techniques: Expressing, Modification, Sequencing, Two Tailed Test

Journal: bioRxiv

Article Title: Trans-kingdom coupling of redox signaling to environmental cell stress responses through multiphase partitioning

doi: 10.1101/2025.05.09.653213

Figure Lengend Snippet: (A) Biotin switch immunoprecipitation and immunoblot with anti-RACK1 showing increased proportion of oxidized RACK1 following IL-1β (10 ng/μL) in WT versus NOX1 knockout colonic epithelial cells. (B) NF-κB activity in HEK cells expressing scrambled vector (EV), or knockdown of RACK1 (siRNA) following IL-1β (10 ng/μL). ***p < 0.001

Article Snippet: The pEGFP-N1-Rack1 plasmid was obtained from Addgene (#41088).

Techniques: Immunoprecipitation, Western Blot, Knock-Out, Activity Assay, Expressing, Plasmid Preparation, Knockdown

Journal: bioRxiv

Article Title: Trans-kingdom coupling of redox signaling to environmental cell stress responses through multiphase partitioning

doi: 10.1101/2025.05.09.653213

Figure Lengend Snippet: (A) NF-κB activity in HEK cells stimulated with IL-1β (10 ng/μL) or with control stimulation with pretreatment with either media, NAC (5mM) or 1mM Rotenone (1mM). (B) NF-κB activity in HEK cells transfected with RACK1-WT, one of the cysteine mutants or empty vector (ev) stimulated with IL-1β (10 ng/ml) or with control. (C) NF-κB activity in HEK transfected with RACK1-WT, C286A mutant or empty vector (ev)with varying dose of IL-1β. (D) p65 immunofluorescence staining in empty vector, RACK1-WT or RACK1-C286A transfected HEK cells either with mock or IL-1β (10 ng/ml) stimulation for 30 minutes. Nuclei was stained using Hoescht stain. (F) Quantification of immunofluorescence image from (D). (G) Western blot graph showing relative p65 phosphorylation in HEK293T expressing either empty vector (ev) (H) Time series of co-immunoprecipitation of IKKα with overexpressed wtRACK1 in HEK293T following IL-1β (10 ng/μL) stimulation. (I) Co-immunoprecipitation of IKKα with RACK1 in HEK293T expressing empty vector (ev), wt or cysteine mutants RACK1 following IL-1β (10 ng/μL,1h) stimulation. (J) NF-κB activity in HEK cells expressing empty vector (ev), wt or cysteine mutants RACK1 stimulated with TNFα, LPS or unstimulated. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The pEGFP-N1-Rack1 plasmid was obtained from Addgene (#41088).

Techniques: Activity Assay, Control, Transfection, Plasmid Preparation, Mutagenesis, Immunofluorescence, Staining, Western Blot, Phospho-proteomics, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: Trans-kingdom coupling of redox signaling to environmental cell stress responses through multiphase partitioning

doi: 10.1101/2025.05.09.653213

Figure Lengend Snippet: (A) Representative fluorescence image showing RACK1 condensates in HCT-8 cells expressing RACK1-GFP and stimulated with IL-1β (10 ng/μL) or with 17-AAG (10 µM) but not in untreated cells. (B) Representative fluorescence image showing time course of RACK1 condensate formation in HCT-8 cells expressing RACK1-GFP stimulated with IL-1β (10 ng/μL). (C) Fluorescence recovery after after photobleaching (FRAP) time course of RACK1-GFP condensates after IL-1β (10 ng/μL) stimulation(orange) and unstimulated cytosolic RACK1-GFP(black). The solid line represents the mean and the dash line represents the error bounds defined by the standard deviation. (D) Representative fluorescence image showing colocalization of RACK1-GFP and INAVA-mcherry in HCT-8 cells upon stimulation with IL-1β (10 ng/μL) or with 17-AAG (10 µM). (E) Pearson correlation of coefficient to quantify colocalization between RACK1-GFP and INAVA-mcherry in HCT-8 cells upon stimulation with IL-1β (10 ng/μL) or with 17-AAG (10 µM). (F) Representative Co-immunoprecipitation blots showing interaction between RACK1 and GFP at two different timepoints in unstimulated or when stimulated with IL-1β (10 ng/μL) or with 17-AAG (10 µM) (G) Quantification of (F). (H) Representative fluorescence image of Caco-2 cells, untreated or stimulated with IL-1β (10 ng/μL) and/or Nox1 inhibitor for 1 hour and stained with DCP-Rho1(10 μM). (K) Representative ratiometric fluorescence image of HCT-8 cells overexpressing RACK1-mcherry. The intensity is the ratio of fluorescence signals excited by 488 nm and 405 nm lasers. (L) Quantification of intensity ratio described in (K) as a function of time. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The pEGFP-N1-Rack1 plasmid was obtained from Addgene (#41088).

Techniques: Fluorescence, Expressing, Standard Deviation, Immunoprecipitation, Staining

Journal: bioRxiv

Article Title: Trans-kingdom coupling of redox signaling to environmental cell stress responses through multiphase partitioning

doi: 10.1101/2025.05.09.653213

Figure Lengend Snippet: (A) PDB structure of RACK1 almost no unstructured regions (B) PDB structure of INAVA as predicted by Alphafold showing large regions of highly unstructured regions and low -residue model confidence score. (C) Predicted Disorder score of RACK1 with and without changes in redox state of amino acids in protein.( , ) (D) Predicted Disorder score of INAVA with and without changes in redox state of amino acids in protein. Representative immunofluorescence image of HCT-8 overexpressing wt RACK1-mcherry and stained with G3BP and stimulated with 17AAG (10 µM) (E) and (F) IL-1β (10 ng/μL). (G) Pearson correlation coefficient showing colocalization of G3BP and Rack1. (E) shows a representative example with a cell with high colocalization and (F) represents a cell with low colocalization.

Article Snippet: The pEGFP-N1-Rack1 plasmid was obtained from Addgene (#41088).

Techniques: Residue, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Trans-kingdom coupling of redox signaling to environmental cell stress responses through multiphase partitioning

doi: 10.1101/2025.05.09.653213

Figure Lengend Snippet: (A) Evolutionary distance of different eukaryotic RACK1 (B) Sequence alignment of 7WD domains of human RACK1. (C) RACK1 homologs across diverse kingdoms as a sunburst plot. (D) Sequence alignment of different bacterial homologs (E) WD7 sequence alignment between human and Entamoeba histolytica RACK1 showing conserved cysteine.

Article Snippet: The pEGFP-N1-Rack1 plasmid was obtained from Addgene (#41088).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Trans-kingdom coupling of redox signaling to environmental cell stress responses through multiphase partitioning

doi: 10.1101/2025.05.09.653213

Figure Lengend Snippet: (A) Multiple Sequence alignment of amino acids proximal to cysteine 286 in eukaryotes. (B) Representative immunofluorescence image of fission yeast cells expressing Flag-cpc2 and treated with either 10% ethanol or media control for 10 minutes. (C) Quantification of number of puncta per cell in control and ethanol treated yeast. (D) Multiple sequence alignment of the amino acids proximal to cysteine 286 in human RACK1 against bacterial homologs. (E) Representative ratiometric fluorescence image of E. coli expressing bacterial analog of RACK1 tagged to Hyper3. (F) Quantification of intensity ratio of E. coli expressing bacterial analog of RACK1 tagged to Hyper3 when treated with either 10% ethanol or media control. (G) Representative fluorescence image of E. coli expressing human RACK1 protein tagged with GFP treated with either 10% ethanol or media control for 10 minutes. (H) Quantification of normalized coefficient of variance in GFP signal as a measure of heterogeneity of RACK1 signal in the cytosol. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The pEGFP-N1-Rack1 plasmid was obtained from Addgene (#41088).

Techniques: Sequencing, Immunofluorescence, Expressing, Control, Fluorescence

Journal: bioRxiv

Article Title: Trans-kingdom coupling of redox signaling to environmental cell stress responses through multiphase partitioning

doi: 10.1101/2025.05.09.653213

Figure Lengend Snippet: (A) String network analysis of redox-proteomics screen colored by manually selected subset of significant functional module. (B) Representative flow cytometry data showing intensity of puromycin incorporation in HCT-8 cells expressing either Rack-1 WT mCherry (top) or Rack-1 Call mCherry (bottom) treated with 17-AAG (10 µM, 10 minutes, orange curve) or untreated (blue). (C) Puromycin incorporation in HCT-8 cells expressing either Rack-1 WT mCherry or Rack-1 Call mCherry treated with 17-AAG (10 µM, 10 minutes) normalized to untreated. (D) NF-κB activity in HEK cells overexpressing empty vector, RACK1-WT or RACK1-C all stimulated with LPS (10 mg/ml). (E) Representative fluorescence image showing IRF3 translocation in HEK cells overexpressing empty vector, RACK1-WT or RACK1-Call stimulated with poly I:C (10µg/ml) (F) IP10 production following infection of HEK293T cells overexpressing empty vector, RACK1-WT, RACK1-C286A or RACK1-Call mutant with SeV (MOI = 1, 1h) (G) Representative ratiometric fluorescence image of Human duodenal organoids transfected with Rack1-Hyper3 and exposed to E. coli strains—pathogenic EPEC O127:H6, non-pathogenic, or uninfected. Quantification of number of puncta formed (H) and Mean Puncta Intensity (I) per transfected organoid. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The pEGFP-N1-Rack1 plasmid was obtained from Addgene (#41088).

Techniques: Functional Assay, Flow Cytometry, Expressing, Activity Assay, Plasmid Preparation, Fluorescence, Translocation Assay, Infection, Mutagenesis, Transfection

(A) Mass spectrometry analysis of NLRP3, RACK1, and NEK7 peptides after purification of NLRP3 complexes. (B and C) Tagged NLRP3 (NLRP3-SFP) or NEK7 (NEK7-SFP) was immunoprecipitated (IP) with anti-FLAG antibody from iBMDM treated with LPS (200 ng Ml−1, 4 h) alone or with LPS plus ATP (5 mM, 30 min) and was immunoblotted with the indicated antibodies. EV, empty vector. (D) BMDM was stimulated with LPS (200 ng mL−1, 4 h) alone or with LPS plus ATP (5 mM, 30 min). Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (E) NLRP3-SFP was co-expressed with HA-tagged RACK1 in HEK293T cells, pulled down, and analyzed by immunoblotting. HA, hemagglutinin. (F) FLAG-tagged, full-length or truncated NLRP3 was co-expressed with HA-tagged RACK1 in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting. (Δpyrin, pyrin domain deleted; ΔLRR, leucine-rich repeats deleted; pyrin, pyrin domain only; NOD, NOD domain only; LRR, leucine-rich repeat only). Results are representative of three independent experiments.

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A) Mass spectrometry analysis of NLRP3, RACK1, and NEK7 peptides after purification of NLRP3 complexes. (B and C) Tagged NLRP3 (NLRP3-SFP) or NEK7 (NEK7-SFP) was immunoprecipitated (IP) with anti-FLAG antibody from iBMDM treated with LPS (200 ng Ml−1, 4 h) alone or with LPS plus ATP (5 mM, 30 min) and was immunoblotted with the indicated antibodies. EV, empty vector. (D) BMDM was stimulated with LPS (200 ng mL−1, 4 h) alone or with LPS plus ATP (5 mM, 30 min). Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (E) NLRP3-SFP was co-expressed with HA-tagged RACK1 in HEK293T cells, pulled down, and analyzed by immunoblotting. HA, hemagglutinin. (F) FLAG-tagged, full-length or truncated NLRP3 was co-expressed with HA-tagged RACK1 in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting. (Δpyrin, pyrin domain deleted; ΔLRR, leucine-rich repeats deleted; pyrin, pyrin domain only; NOD, NOD domain only; LRR, leucine-rich repeat only). Results are representative of three independent experiments.

Article Snippet: RACK1 cDNA plasmid (pEGFP-N1-RACK1) , Addgene , Cat# 41088.

Techniques: Mass Spectrometry, Purification, Immunoprecipitation, Plasmid Preparation, Western Blot

(A–C) iBMDM was treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). The cells were left unstimulated (PBS) or stimulated with LPS (200 ng mL−1, 4 h), ATP (5 mM, 30 min), or LPS (200 ng mL−1, 4 h) plus ATP (5 mM, 30 min). (A) Supernatants (Sup.) and cell lysates (Lys.) were analyzed by immunoblotting with the indicated antibodies. (B and C) IL-1β and TNF-α release in supernatants was analyzed by ELISA. (D–G) iBMDM were treated with siControl or siRACK1 and then primed by LPS (200 ng mL−1, 4 h). (B–G) Cells were left unstimulated (PBS) or stimulated by nigericin (5 μM, 1 h), gramicidin (0.5 μM, 1 h), LLOMe (2 μM, 4 h), silica (500 μg Ml−1, 4 h), poly(dA:dT) (2 μg mL−1, 4 h), or Salmonella (MOI = 10, 1 h). Caspase-1 (Casp1) in the supernatant (Sup.) and cell lysate (Lys.) were analyzed by immunoblotting (D and G); IL-1β and TNF-α release in the supernatants of (D) was analyzed by ELISA (E and F). (H–J) Mice were intraperitoneally injected with in vivo-jetPEI-encapsulated siRNA (100 μg per mouse) and then with LPS (25 mg/kg of body weight). Mouse serum was collected after intraperitoneal injection of LPS and analyzed for cytokines IL-1β (I) and TNF-α (J). Peritoneal exudate cells (PECs) from each mouse were collected for assessing RACK1 expression by western blot (H). Actin was immunoblotted as a loading control. Each symbol represents one mouse. For ELISA data of in vitro experiments (B, C, E, and F), error bars denote SD of triplicate wells. Results (A–G) are representative of three independent experiments. For in vivo experiments (H–J), mean values are indicated by a horizontal bar. Results are representative of two independent experiments. NS, not significant. Unpaired two-tailed Student’s t test, *p < 0.05.

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A–C) iBMDM was treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). The cells were left unstimulated (PBS) or stimulated with LPS (200 ng mL−1, 4 h), ATP (5 mM, 30 min), or LPS (200 ng mL−1, 4 h) plus ATP (5 mM, 30 min). (A) Supernatants (Sup.) and cell lysates (Lys.) were analyzed by immunoblotting with the indicated antibodies. (B and C) IL-1β and TNF-α release in supernatants was analyzed by ELISA. (D–G) iBMDM were treated with siControl or siRACK1 and then primed by LPS (200 ng mL−1, 4 h). (B–G) Cells were left unstimulated (PBS) or stimulated by nigericin (5 μM, 1 h), gramicidin (0.5 μM, 1 h), LLOMe (2 μM, 4 h), silica (500 μg Ml−1, 4 h), poly(dA:dT) (2 μg mL−1, 4 h), or Salmonella (MOI = 10, 1 h). Caspase-1 (Casp1) in the supernatant (Sup.) and cell lysate (Lys.) were analyzed by immunoblotting (D and G); IL-1β and TNF-α release in the supernatants of (D) was analyzed by ELISA (E and F). (H–J) Mice were intraperitoneally injected with in vivo-jetPEI-encapsulated siRNA (100 μg per mouse) and then with LPS (25 mg/kg of body weight). Mouse serum was collected after intraperitoneal injection of LPS and analyzed for cytokines IL-1β (I) and TNF-α (J). Peritoneal exudate cells (PECs) from each mouse were collected for assessing RACK1 expression by western blot (H). Actin was immunoblotted as a loading control. Each symbol represents one mouse. For ELISA data of in vitro experiments (B, C, E, and F), error bars denote SD of triplicate wells. Results (A–G) are representative of three independent experiments. For in vivo experiments (H–J), mean values are indicated by a horizontal bar. Results are representative of two independent experiments. NS, not significant. Unpaired two-tailed Student’s t test, *p < 0.05.

Article Snippet: RACK1 cDNA plasmid (pEGFP-N1-RACK1) , Addgene , Cat# 41088.

Techniques: Control, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, In Vivo, Expressing, In Vitro, Two Tailed Test

(A–E) Macrophages (NLRP3 R258W) were treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). Cells were left unstimulated (PBS) or stimulated with LPS (200 ng mL−1, 4 h). Cell lysates (Lys.) and culture supernatants (Sup.) were immunoblotted with indicated antibodies (A and D). IL-1β (B), TNF-α (C), and LDH(E) in culture supernatants of macrophages were analyzed. Error bars denote SD of triplicate wells. Results are representative of three independent experiments. Unpaired two-tailed Student’s t test, *p < 0.05.

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A–E) Macrophages (NLRP3 R258W) were treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). Cells were left unstimulated (PBS) or stimulated with LPS (200 ng mL−1, 4 h). Cell lysates (Lys.) and culture supernatants (Sup.) were immunoblotted with indicated antibodies (A and D). IL-1β (B), TNF-α (C), and LDH(E) in culture supernatants of macrophages were analyzed. Error bars denote SD of triplicate wells. Results are representative of three independent experiments. Unpaired two-tailed Student’s t test, *p < 0.05.

Article Snippet: RACK1 cDNA plasmid (pEGFP-N1-RACK1) , Addgene , Cat# 41088.

Techniques: Control, Two Tailed Test

$(A) iBMDM was treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). Cells were primed with LPS (200 ng mL−1, 4 h) and then stimulated with ATP (5 mM, 30 min), nigericin (5 μM, 1 h), or Salmonella (MOI = 10, 1 h). Representative images of ASC specks in macrophages treated with indicated stimuli. Scale bars, 5 μm. (B) Quantification of endogenous ASC specks (arrows) in (A). Data show representative results from three combined independent experiments. Error bars indicate SD. Unpaired two-tailed Student’s t test, *p < 0.05. (C) ASC oligomerization in Triton X-100 (TX)-insoluble fractions of macrophages was analyzed by immunoblotting after cross-linking. TX-soluble fractions were also immunoblotted with indicated antibodies. Results are representative of three independent experiments.$

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A) iBMDM was treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). Cells were primed with LPS (200 ng mL−1, 4 h) and then stimulated with ATP (5 mM, 30 min), nigericin (5 μM, 1 h), or Salmonella (MOI = 10, 1 h). Representative images of ASC specks in macrophages treated with indicated stimuli. Scale bars, 5 μm. (B) Quantification of endogenous ASC specks (arrows) in (A). Data show representative results from three combined independent experiments. Error bars indicate SD. Unpaired two-tailed Student’s t test, *p < 0.05. (C) ASC oligomerization in Triton X-100 (TX)-insoluble fractions of macrophages was analyzed by immunoblotting after cross-linking. TX-soluble fractions were also immunoblotted with indicated antibodies. Results are representative of three independent experiments.

Article Snippet: RACK1 cDNA plasmid (pEGFP-N1-RACK1) , Addgene , Cat# 41088.

Techniques: Control, Two Tailed Test, Western Blot

(A) Tagged NLRP3 (NLRP3-SFP) was co-expressed with HA-tagged wild-type RACK1 or mutant RACK1 (R36D/K38E, defective in ribosome binding) in HEK293T cells. Cell lysates were pulled down with streptavidin beads and analyzed by immunoblotting. (B–D) Mouse macrophages were treated with RACK1 siRNA (siRACK1), followed by transduction with lentivirus expressing HA-tagged, siRNA-resistant, wild-type RACK1 or mutant RACK1 (R36D/K38E). Macrophages were primed with LPS (200 ng mL−1, 4 h) and then stimulated with PBS (control), ATP (5 mM, 30 min), or nigericin (5 μM, 1 h). Mixtures of cell lysates and culture supernatants were analyzed by immunoblotting with the indicated antibodies (B). IL-1β (C) and TNF-α (D) in culture supernatants were analyzed by ELISA. Error bars denote SD of triplicate wells. Results are representative of three independent experiments. Unpaired two-tailed Student’s t test, *p < 0.05.

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A) Tagged NLRP3 (NLRP3-SFP) was co-expressed with HA-tagged wild-type RACK1 or mutant RACK1 (R36D/K38E, defective in ribosome binding) in HEK293T cells. Cell lysates were pulled down with streptavidin beads and analyzed by immunoblotting. (B–D) Mouse macrophages were treated with RACK1 siRNA (siRACK1), followed by transduction with lentivirus expressing HA-tagged, siRNA-resistant, wild-type RACK1 or mutant RACK1 (R36D/K38E). Macrophages were primed with LPS (200 ng mL−1, 4 h) and then stimulated with PBS (control), ATP (5 mM, 30 min), or nigericin (5 μM, 1 h). Mixtures of cell lysates and culture supernatants were analyzed by immunoblotting with the indicated antibodies (B). IL-1β (C) and TNF-α (D) in culture supernatants were analyzed by ELISA. Error bars denote SD of triplicate wells. Results are representative of three independent experiments. Unpaired two-tailed Student’s t test, *p < 0.05.

Article Snippet: RACK1 cDNA plasmid (pEGFP-N1-RACK1) , Addgene , Cat# 41088.

Techniques: Mutagenesis, Binding Assay, Western Blot, Transduction, Expressing, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

(A) Macrophages were infected with lentivirus expressing control shRNA (shControl) or RACK1 shRNA (shRACK1). After puromycin selection, transduced macrophages were primed with LPS (200 ng mL−1, 4 h) and then stimulated with PBS (control), ATP (5 mM, 30 min), or nigericin (5 μM, 1 h). NLRP3 inflammasome assembly was analyzed by blue native PAGE and immunoblotting. Cell lysates were also analyzed by SDS–PAGE and immunoblotting. (B) Cell lysates were separated by a first dimension of blue native PAGE, followed by a second dimension of SDS–PAGE. (C) HEK293T cells stably expressing YFP-NLRP3-Luc were transfected with 200 nM of control siRNA (siControl) or the indicated concentrations of RACK1 siRNA (siRACK1). The relative levels of RACK1 gene expression were analyzed by quantitative reverse transcriptase-PCR and normalized to the level of endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. (D) Intramolecular BRET signal of NLRP3 was measured before and after nigericin (10 μM) treatment. The arrow indicates the time for nigericin addition. Mann-Whitney test, two-tailed for each comparison of siControl (200 nM) with siRACK1 (200 nM). *p < 0.05. Results are representative of three independent experiments.

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A) Macrophages were infected with lentivirus expressing control shRNA (shControl) or RACK1 shRNA (shRACK1). After puromycin selection, transduced macrophages were primed with LPS (200 ng mL−1, 4 h) and then stimulated with PBS (control), ATP (5 mM, 30 min), or nigericin (5 μM, 1 h). NLRP3 inflammasome assembly was analyzed by blue native PAGE and immunoblotting. Cell lysates were also analyzed by SDS–PAGE and immunoblotting. (B) Cell lysates were separated by a first dimension of blue native PAGE, followed by a second dimension of SDS–PAGE. (C) HEK293T cells stably expressing YFP-NLRP3-Luc were transfected with 200 nM of control siRNA (siControl) or the indicated concentrations of RACK1 siRNA (siRACK1). The relative levels of RACK1 gene expression were analyzed by quantitative reverse transcriptase-PCR and normalized to the level of endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. (D) Intramolecular BRET signal of NLRP3 was measured before and after nigericin (10 μM) treatment. The arrow indicates the time for nigericin addition. Mann-Whitney test, two-tailed for each comparison of siControl (200 nM) with siRACK1 (200 nM). *p < 0.05. Results are representative of three independent experiments.

Article Snippet: RACK1 cDNA plasmid (pEGFP-N1-RACK1) , Addgene , Cat# 41088.

Techniques: Infection, Expressing, Control, shRNA, Selection, Blue Native PAGE, Western Blot, SDS Page, Stable Transfection, Transfection, Gene Expression, Reverse Transcription, MANN-WHITNEY, Two Tailed Test, Comparison

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: RACK1 cDNA plasmid (pEGFP-N1-RACK1) , Addgene , Cat# 41088.

Techniques: Control, Virus, Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Mutagenesis, cDNA Synthesis, In Vivo, Plasmid Preparation, Software, Membrane

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