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A) SW480 cells with GJB3 knockdown were analyzed by RNA sequencing. Differentially expressed genes (upregulated and downregulated) were identified and their normalized expression values (Z-score transformed Transcripts Per Million - TPM) are visualized as a heatmap. B) Gene ontology (GO) analysis was performed using Metascape software on the differentially expressed genes identified in GJB3 knockdown cells. The most significantly enriched GO terms are presented. C) GO analysis was performed specifically on the upregulated genes identified in GJB3 knockdown cells. The top enriched terms are displayed in a bubble plot. D) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 24 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. E) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 48 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. F) Expression level of ATF target as determine by RNA-seq data in SW480 cells after treatment with control or shGJB3 plasmids. G) SW480 cells stably expressing both <t>GFP-LC3</t> and <t>RFP-LC3ΔG</t> were used to assess the effect of GJB3 knockdown on autophagy. Cells were transfected with two different shRNAs targeting GJB3 or a control vector. Knockdown efficiency was confirmed, and then cells were imaged for GFP and RFP fluorescence. H) SW480 cells were used for GJB3 knockdown using control and GJB3 shRNAs. After knockdown the cells were stained with a caspase-3 activity detection reagent, and fluorescence intensity was measured at given time points. The line plot shown to detect the level of apoptosis of different cells. I) Control and GJB3 knockdown SW480 cells were transfected with siATG5 or treated with 3-methyladenine (3-MA), an inhibitor of autophagy. Protein lysates were then extracted and analyzed by Western blotting to assess LC3 protein levels. J) Control and GJB3 knockdown SW480 cells were plated, and their proliferation was monitored using an Incucyte live-cell imaging system. K) Control and GJB3 knockdown SW480 cells were transfected with siATG5, and then plated for proliferation analysis using the Incucyte live-cell imaging system. L) Control and GJB3 knockdown SW480 cells were treated with 3-MA and plated. Proliferation was measured using the Incucyte live-cell imaging system.
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A) SW480 cells with GJB3 knockdown were analyzed by RNA sequencing. Differentially expressed genes (upregulated and downregulated) were identified and their normalized expression values (Z-score transformed Transcripts Per Million - TPM) are visualized as a heatmap. B) Gene ontology (GO) analysis was performed using Metascape software on the differentially expressed genes identified in GJB3 knockdown cells. The most significantly enriched GO terms are presented. C) GO analysis was performed specifically on the upregulated genes identified in GJB3 knockdown cells. The top enriched terms are displayed in a bubble plot. D) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 24 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. E) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 48 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. F) Expression level of ATF target as determine by RNA-seq data in SW480 cells after treatment with control or shGJB3 plasmids. G) SW480 cells stably expressing both <t>GFP-LC3</t> and <t>RFP-LC3ΔG</t> were used to assess the effect of GJB3 knockdown on autophagy. Cells were transfected with two different shRNAs targeting GJB3 or a control vector. Knockdown efficiency was confirmed, and then cells were imaged for GFP and RFP fluorescence. H) SW480 cells were used for GJB3 knockdown using control and GJB3 shRNAs. After knockdown the cells were stained with a caspase-3 activity detection reagent, and fluorescence intensity was measured at given time points. The line plot shown to detect the level of apoptosis of different cells. I) Control and GJB3 knockdown SW480 cells were transfected with siATG5 or treated with 3-methyladenine (3-MA), an inhibitor of autophagy. Protein lysates were then extracted and analyzed by Western blotting to assess LC3 protein levels. J) Control and GJB3 knockdown SW480 cells were plated, and their proliferation was monitored using an Incucyte live-cell imaging system. K) Control and GJB3 knockdown SW480 cells were transfected with siATG5, and then plated for proliferation analysis using the Incucyte live-cell imaging system. L) Control and GJB3 knockdown SW480 cells were treated with 3-MA and plated. Proliferation was measured using the Incucyte live-cell imaging system.
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A) SW480 cells with GJB3 knockdown were analyzed by RNA sequencing. Differentially expressed genes (upregulated and downregulated) were identified and their normalized expression values (Z-score transformed Transcripts Per Million - TPM) are visualized as a heatmap. B) Gene ontology (GO) analysis was performed using Metascape software on the differentially expressed genes identified in GJB3 knockdown cells. The most significantly enriched GO terms are presented. C) GO analysis was performed specifically on the upregulated genes identified in GJB3 knockdown cells. The top enriched terms are displayed in a bubble plot. D) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 24 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. E) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 48 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. F) Expression level of ATF target as determine by RNA-seq data in SW480 cells after treatment with control or shGJB3 plasmids. G) SW480 cells stably expressing both <t>GFP-LC3</t> and <t>RFP-LC3ΔG</t> were used to assess the effect of GJB3 knockdown on autophagy. Cells were transfected with two different shRNAs targeting GJB3 or a control vector. Knockdown efficiency was confirmed, and then cells were imaged for GFP and RFP fluorescence. H) SW480 cells were used for GJB3 knockdown using control and GJB3 shRNAs. After knockdown the cells were stained with a caspase-3 activity detection reagent, and fluorescence intensity was measured at given time points. The line plot shown to detect the level of apoptosis of different cells. I) Control and GJB3 knockdown SW480 cells were transfected with siATG5 or treated with 3-methyladenine (3-MA), an inhibitor of autophagy. Protein lysates were then extracted and analyzed by Western blotting to assess LC3 protein levels. J) Control and GJB3 knockdown SW480 cells were plated, and their proliferation was monitored using an Incucyte live-cell imaging system. K) Control and GJB3 knockdown SW480 cells were transfected with siATG5, and then plated for proliferation analysis using the Incucyte live-cell imaging system. L) Control and GJB3 knockdown SW480 cells were treated with 3-MA and plated. Proliferation was measured using the Incucyte live-cell imaging system.
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gfp lc3 rfp lc3dg autophagy flux reporter assay pmrx ip gfp lc3 rfp lc3dg  (Addgene inc)
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A) SW480 cells with GJB3 knockdown were analyzed by RNA sequencing. Differentially expressed genes (upregulated and downregulated) were identified and their normalized expression values (Z-score transformed Transcripts Per Million - TPM) are visualized as a heatmap. B) Gene ontology (GO) analysis was performed using Metascape software on the differentially expressed genes identified in GJB3 knockdown cells. The most significantly enriched GO terms are presented. C) GO analysis was performed specifically on the upregulated genes identified in GJB3 knockdown cells. The top enriched terms are displayed in a bubble plot. D) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 24 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. E) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 48 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. F) Expression level of ATF target as determine by RNA-seq data in SW480 cells after treatment with control or shGJB3 plasmids. G) SW480 cells stably expressing both <t>GFP-LC3</t> and <t>RFP-LC3ΔG</t> were used to assess the effect of GJB3 knockdown on autophagy. Cells were transfected with two different shRNAs targeting GJB3 or a control vector. Knockdown efficiency was confirmed, and then cells were imaged for GFP and RFP fluorescence. H) SW480 cells were used for GJB3 knockdown using control and GJB3 shRNAs. After knockdown the cells were stained with a caspase-3 activity detection reagent, and fluorescence intensity was measured at given time points. The line plot shown to detect the level of apoptosis of different cells. I) Control and GJB3 knockdown SW480 cells were transfected with siATG5 or treated with 3-methyladenine (3-MA), an inhibitor of autophagy. Protein lysates were then extracted and analyzed by Western blotting to assess LC3 protein levels. J) Control and GJB3 knockdown SW480 cells were plated, and their proliferation was monitored using an Incucyte live-cell imaging system. K) Control and GJB3 knockdown SW480 cells were transfected with siATG5, and then plated for proliferation analysis using the Incucyte live-cell imaging system. L) Control and GJB3 knockdown SW480 cells were treated with 3-MA and plated. Proliferation was measured using the Incucyte live-cell imaging system.
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fuw mcherry gfp lc3 reporter plasmid  (Addgene inc)
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A) SW480 cells with GJB3 knockdown were analyzed by RNA sequencing. Differentially expressed genes (upregulated and downregulated) were identified and their normalized expression values (Z-score transformed Transcripts Per Million - TPM) are visualized as a heatmap. B) Gene ontology (GO) analysis was performed using Metascape software on the differentially expressed genes identified in GJB3 knockdown cells. The most significantly enriched GO terms are presented. C) GO analysis was performed specifically on the upregulated genes identified in GJB3 knockdown cells. The top enriched terms are displayed in a bubble plot. D) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 24 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. E) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 48 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. F) Expression level of ATF target as determine by RNA-seq data in SW480 cells after treatment with control or shGJB3 plasmids. G) SW480 cells stably expressing both <t>GFP-LC3</t> and <t>RFP-LC3ΔG</t> were used to assess the effect of GJB3 knockdown on autophagy. Cells were transfected with two different shRNAs targeting GJB3 or a control vector. Knockdown efficiency was confirmed, and then cells were imaged for GFP and RFP fluorescence. H) SW480 cells were used for GJB3 knockdown using control and GJB3 shRNAs. After knockdown the cells were stained with a caspase-3 activity detection reagent, and fluorescence intensity was measured at given time points. The line plot shown to detect the level of apoptosis of different cells. I) Control and GJB3 knockdown SW480 cells were transfected with siATG5 or treated with 3-methyladenine (3-MA), an inhibitor of autophagy. Protein lysates were then extracted and analyzed by Western blotting to assess LC3 protein levels. J) Control and GJB3 knockdown SW480 cells were plated, and their proliferation was monitored using an Incucyte live-cell imaging system. K) Control and GJB3 knockdown SW480 cells were transfected with siATG5, and then plated for proliferation analysis using the Incucyte live-cell imaging system. L) Control and GJB3 knockdown SW480 cells were treated with 3-MA and plated. Proliferation was measured using the Incucyte live-cell imaging system.
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mcherry-gfp-lc3 reporter plasmid c3011  (Beyotime)
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A) SW480 cells with GJB3 knockdown were analyzed by RNA sequencing. Differentially expressed genes (upregulated and downregulated) were identified and their normalized expression values (Z-score transformed Transcripts Per Million - TPM) are visualized as a heatmap. B) Gene ontology (GO) analysis was performed using Metascape software on the differentially expressed genes identified in GJB3 knockdown cells. The most significantly enriched GO terms are presented. C) GO analysis was performed specifically on the upregulated genes identified in GJB3 knockdown cells. The top enriched terms are displayed in a bubble plot. D) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 24 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. E) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 48 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. F) Expression level of ATF target as determine by RNA-seq data in SW480 cells after treatment with control or shGJB3 plasmids. G) SW480 cells stably expressing both <t>GFP-LC3</t> and <t>RFP-LC3ΔG</t> were used to assess the effect of GJB3 knockdown on autophagy. Cells were transfected with two different shRNAs targeting GJB3 or a control vector. Knockdown efficiency was confirmed, and then cells were imaged for GFP and RFP fluorescence. H) SW480 cells were used for GJB3 knockdown using control and GJB3 shRNAs. After knockdown the cells were stained with a caspase-3 activity detection reagent, and fluorescence intensity was measured at given time points. The line plot shown to detect the level of apoptosis of different cells. I) Control and GJB3 knockdown SW480 cells were transfected with siATG5 or treated with 3-methyladenine (3-MA), an inhibitor of autophagy. Protein lysates were then extracted and analyzed by Western blotting to assess LC3 protein levels. J) Control and GJB3 knockdown SW480 cells were plated, and their proliferation was monitored using an Incucyte live-cell imaging system. K) Control and GJB3 knockdown SW480 cells were transfected with siATG5, and then plated for proliferation analysis using the Incucyte live-cell imaging system. L) Control and GJB3 knockdown SW480 cells were treated with 3-MA and plated. Proliferation was measured using the Incucyte live-cell imaging system.
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A) SW480 cells with GJB3 knockdown were analyzed by RNA sequencing. Differentially expressed genes (upregulated and downregulated) were identified and their normalized expression values (Z-score transformed Transcripts Per Million - TPM) are visualized as a heatmap. B) Gene ontology (GO) analysis was performed using Metascape software on the differentially expressed genes identified in GJB3 knockdown cells. The most significantly enriched GO terms are presented. C) GO analysis was performed specifically on the upregulated genes identified in GJB3 knockdown cells. The top enriched terms are displayed in a bubble plot. D) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 24 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. E) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 48 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. F) Expression level of ATF target as determine by RNA-seq data in SW480 cells after treatment with control or shGJB3 plasmids. G) SW480 cells stably expressing both GFP-LC3 and RFP-LC3ΔG were used to assess the effect of GJB3 knockdown on autophagy. Cells were transfected with two different shRNAs targeting GJB3 or a control vector. Knockdown efficiency was confirmed, and then cells were imaged for GFP and RFP fluorescence. H) SW480 cells were used for GJB3 knockdown using control and GJB3 shRNAs. After knockdown the cells were stained with a caspase-3 activity detection reagent, and fluorescence intensity was measured at given time points. The line plot shown to detect the level of apoptosis of different cells. I) Control and GJB3 knockdown SW480 cells were transfected with siATG5 or treated with 3-methyladenine (3-MA), an inhibitor of autophagy. Protein lysates were then extracted and analyzed by Western blotting to assess LC3 protein levels. J) Control and GJB3 knockdown SW480 cells were plated, and their proliferation was monitored using an Incucyte live-cell imaging system. K) Control and GJB3 knockdown SW480 cells were transfected with siATG5, and then plated for proliferation analysis using the Incucyte live-cell imaging system. L) Control and GJB3 knockdown SW480 cells were treated with 3-MA and plated. Proliferation was measured using the Incucyte live-cell imaging system.

Journal: bioRxiv

Article Title: GJB3, a gap junction gene, supports cell growth by mediating cystine uptake and regulating cellular stress pathways in SLC7A11 low adenocarcinomas

doi: 10.1101/2024.12.03.626548

Figure Lengend Snippet: A) SW480 cells with GJB3 knockdown were analyzed by RNA sequencing. Differentially expressed genes (upregulated and downregulated) were identified and their normalized expression values (Z-score transformed Transcripts Per Million - TPM) are visualized as a heatmap. B) Gene ontology (GO) analysis was performed using Metascape software on the differentially expressed genes identified in GJB3 knockdown cells. The most significantly enriched GO terms are presented. C) GO analysis was performed specifically on the upregulated genes identified in GJB3 knockdown cells. The top enriched terms are displayed in a bubble plot. D) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 24 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. E) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 48 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. F) Expression level of ATF target as determine by RNA-seq data in SW480 cells after treatment with control or shGJB3 plasmids. G) SW480 cells stably expressing both GFP-LC3 and RFP-LC3ΔG were used to assess the effect of GJB3 knockdown on autophagy. Cells were transfected with two different shRNAs targeting GJB3 or a control vector. Knockdown efficiency was confirmed, and then cells were imaged for GFP and RFP fluorescence. H) SW480 cells were used for GJB3 knockdown using control and GJB3 shRNAs. After knockdown the cells were stained with a caspase-3 activity detection reagent, and fluorescence intensity was measured at given time points. The line plot shown to detect the level of apoptosis of different cells. I) Control and GJB3 knockdown SW480 cells were transfected with siATG5 or treated with 3-methyladenine (3-MA), an inhibitor of autophagy. Protein lysates were then extracted and analyzed by Western blotting to assess LC3 protein levels. J) Control and GJB3 knockdown SW480 cells were plated, and their proliferation was monitored using an Incucyte live-cell imaging system. K) Control and GJB3 knockdown SW480 cells were transfected with siATG5, and then plated for proliferation analysis using the Incucyte live-cell imaging system. L) Control and GJB3 knockdown SW480 cells were treated with 3-MA and plated. Proliferation was measured using the Incucyte live-cell imaging system.

Article Snippet: To study autophagy in GJB3-silenced cells, the autophagosome reporter plasmid DEST-CMV mCherry-GFP-LC3B WT (Addgene, 123230) and the autophagic flux reporter plasmid pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene, 84572) were employed.

Techniques: Knockdown, RNA Sequencing Assay, Expressing, Transformation Assay, Software, Isolation, Control, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Fluorescence, Staining, Activity Assay, Live Cell Imaging