plasmid pcpg free lucia pcpg lucia Search Results


pcfj151 addgene  (Addgene inc)
90
Addgene inc pcfj151 addgene
Pcfj151 Addgene, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/plasmid+pcpg+free+lucia+pcpg+lucia/10__7554_slash_elife__48119-413-249-250?v=Addgene+inc
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pcfj151 addgene - by Bioz Stars, 2026-08
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pcp1  (ATCC)
90
ATCC pcp1
Pcp1, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pbdkrb2 tcs q g  (Addgene inc)
93
Addgene inc pbdkrb2 tcs q g
Pbdkrb2 Tcs Q G, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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hsp70  (StressMarq)
93
StressMarq hsp70
Bag1 regulates hERG expression. A, diagram of Bag1M transfection constructs used in this study. The UBL and BAG domain boundaries are marked with residue numbers, and the position of the R237A mutation disrupting <t>Hsp70</t> interaction is shown with a star. B, HeLa cells stably expressing hERG were transfected with siRNA against Bag1 (targeting all isoforms) or non-silencing control. CG and FG hERG were detected as 135- and 155-kDa bands, respectively, by immunoblot (IB) and quantified relative to the amount of each in control cells. Averages and standard deviations are shown to the right of data points. Knockdown of Bag1 was confirmed by immunoblot; the bands corresponding to endogenous Bag1L, Bag1M, and Bag1S are marked. C, experiment in B was performed with MG132 treatment, and hERG was quantified relative to MG132-treated non-silencing controls. D, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control. CG and FG hERG were detected and quantified as above. Bands corresponding to Bag1M, Bag1S, and C-Bag1 are marked. Lower panel, the experiment was performed with MG132 treatment and quantitation relative to MG132 treated vector control reported below the blot. E, HEK293 cells were transfected with human transferrin receptor (TfR) and Bag1 or vector control. The receptor was detected by immunoblot, and total amounts were quantified. F, HEK293 cells were transfected with hERG, GFP, and either Bag1 or vector control. Separately, HEK293 cells were transfected with hERG, siGLO, and either siRNA against Bag1 or non-silencing control. Transfected cells were identified by fluorescence microscopy. Voltage response curves from patch clamp measurements are shown. Data points and lines representing the averages are shown. *, p < 0.05; **, p < 0.01 relative to controls.
Hsp70, supplied by StressMarq, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/plasmid+pcpg+free+lucia+pcpg+lucia/pmc05313101-1017-36-37?v=StressMarq
Average 93 stars, based on 1 article reviews
hsp70 - by Bioz Stars, 2026-08
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ubc  (Addgene inc)
92
Addgene inc ubc
Bag1 regulates hERG expression. A, diagram of Bag1M transfection constructs used in this study. The UBL and BAG domain boundaries are marked with residue numbers, and the position of the R237A mutation disrupting <t>Hsp70</t> interaction is shown with a star. B, HeLa cells stably expressing hERG were transfected with siRNA against Bag1 (targeting all isoforms) or non-silencing control. CG and FG hERG were detected as 135- and 155-kDa bands, respectively, by immunoblot (IB) and quantified relative to the amount of each in control cells. Averages and standard deviations are shown to the right of data points. Knockdown of Bag1 was confirmed by immunoblot; the bands corresponding to endogenous Bag1L, Bag1M, and Bag1S are marked. C, experiment in B was performed with MG132 treatment, and hERG was quantified relative to MG132-treated non-silencing controls. D, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control. CG and FG hERG were detected and quantified as above. Bands corresponding to Bag1M, Bag1S, and C-Bag1 are marked. Lower panel, the experiment was performed with MG132 treatment and quantitation relative to MG132 treated vector control reported below the blot. E, HEK293 cells were transfected with human transferrin receptor (TfR) and Bag1 or vector control. The receptor was detected by immunoblot, and total amounts were quantified. F, HEK293 cells were transfected with hERG, GFP, and either Bag1 or vector control. Separately, HEK293 cells were transfected with hERG, siGLO, and either siRNA against Bag1 or non-silencing control. Transfected cells were identified by fluorescence microscopy. Voltage response curves from patch clamp measurements are shown. Data points and lines representing the averages are shown. *, p < 0.05; **, p < 0.01 relative to controls.
Ubc, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/plasmid+pcpg+free+lucia+pcpg+lucia/pmc04992171-222-27-29?v=Addgene+inc
Average 92 stars, based on 1 article reviews
ubc - by Bioz Stars, 2026-08
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jaha 118 010690  (Addgene inc)
91
Addgene inc jaha 118 010690
Bag1 regulates hERG expression. A, diagram of Bag1M transfection constructs used in this study. The UBL and BAG domain boundaries are marked with residue numbers, and the position of the R237A mutation disrupting <t>Hsp70</t> interaction is shown with a star. B, HeLa cells stably expressing hERG were transfected with siRNA against Bag1 (targeting all isoforms) or non-silencing control. CG and FG hERG were detected as 135- and 155-kDa bands, respectively, by immunoblot (IB) and quantified relative to the amount of each in control cells. Averages and standard deviations are shown to the right of data points. Knockdown of Bag1 was confirmed by immunoblot; the bands corresponding to endogenous Bag1L, Bag1M, and Bag1S are marked. C, experiment in B was performed with MG132 treatment, and hERG was quantified relative to MG132-treated non-silencing controls. D, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control. CG and FG hERG were detected and quantified as above. Bands corresponding to Bag1M, Bag1S, and C-Bag1 are marked. Lower panel, the experiment was performed with MG132 treatment and quantitation relative to MG132 treated vector control reported below the blot. E, HEK293 cells were transfected with human transferrin receptor (TfR) and Bag1 or vector control. The receptor was detected by immunoblot, and total amounts were quantified. F, HEK293 cells were transfected with hERG, GFP, and either Bag1 or vector control. Separately, HEK293 cells were transfected with hERG, siGLO, and either siRNA against Bag1 or non-silencing control. Transfected cells were identified by fluorescence microscopy. Voltage response curves from patch clamp measurements are shown. Data points and lines representing the averages are shown. *, p < 0.05; **, p < 0.01 relative to controls.
Jaha 118 010690, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/plasmid+pcpg+free+lucia+pcpg+lucia/10__1161_slash_jaha__118__010690-60-29-13?v=Addgene+inc
Average 91 stars, based on 1 article reviews
jaha 118 010690 - by Bioz Stars, 2026-08
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pcp gfp  (Addgene inc)
93
Addgene inc pcp gfp
Bag1 regulates hERG expression. A, diagram of Bag1M transfection constructs used in this study. The UBL and BAG domain boundaries are marked with residue numbers, and the position of the R237A mutation disrupting <t>Hsp70</t> interaction is shown with a star. B, HeLa cells stably expressing hERG were transfected with siRNA against Bag1 (targeting all isoforms) or non-silencing control. CG and FG hERG were detected as 135- and 155-kDa bands, respectively, by immunoblot (IB) and quantified relative to the amount of each in control cells. Averages and standard deviations are shown to the right of data points. Knockdown of Bag1 was confirmed by immunoblot; the bands corresponding to endogenous Bag1L, Bag1M, and Bag1S are marked. C, experiment in B was performed with MG132 treatment, and hERG was quantified relative to MG132-treated non-silencing controls. D, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control. CG and FG hERG were detected and quantified as above. Bands corresponding to Bag1M, Bag1S, and C-Bag1 are marked. Lower panel, the experiment was performed with MG132 treatment and quantitation relative to MG132 treated vector control reported below the blot. E, HEK293 cells were transfected with human transferrin receptor (TfR) and Bag1 or vector control. The receptor was detected by immunoblot, and total amounts were quantified. F, HEK293 cells were transfected with hERG, GFP, and either Bag1 or vector control. Separately, HEK293 cells were transfected with hERG, siGLO, and either siRNA against Bag1 or non-silencing control. Transfected cells were identified by fluorescence microscopy. Voltage response curves from patch clamp measurements are shown. Data points and lines representing the averages are shown. *, p < 0.05; **, p < 0.01 relative to controls.
Pcp Gfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/plasmid+pcpg+free+lucia+pcpg+lucia/pmc09652286-175-6-7?v=Addgene+inc
Average 93 stars, based on 1 article reviews
pcp gfp - by Bioz Stars, 2026-08
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sc 391691 pcp biotin jena bioscience  (Jena Bioscience)
95
Jena Bioscience sc 391691 pcp biotin jena bioscience
Bag1 regulates hERG expression. A, diagram of Bag1M transfection constructs used in this study. The UBL and BAG domain boundaries are marked with residue numbers, and the position of the R237A mutation disrupting <t>Hsp70</t> interaction is shown with a star. B, HeLa cells stably expressing hERG were transfected with siRNA against Bag1 (targeting all isoforms) or non-silencing control. CG and FG hERG were detected as 135- and 155-kDa bands, respectively, by immunoblot (IB) and quantified relative to the amount of each in control cells. Averages and standard deviations are shown to the right of data points. Knockdown of Bag1 was confirmed by immunoblot; the bands corresponding to endogenous Bag1L, Bag1M, and Bag1S are marked. C, experiment in B was performed with MG132 treatment, and hERG was quantified relative to MG132-treated non-silencing controls. D, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control. CG and FG hERG were detected and quantified as above. Bands corresponding to Bag1M, Bag1S, and C-Bag1 are marked. Lower panel, the experiment was performed with MG132 treatment and quantitation relative to MG132 treated vector control reported below the blot. E, HEK293 cells were transfected with human transferrin receptor (TfR) and Bag1 or vector control. The receptor was detected by immunoblot, and total amounts were quantified. F, HEK293 cells were transfected with hERG, GFP, and either Bag1 or vector control. Separately, HEK293 cells were transfected with hERG, siGLO, and either siRNA against Bag1 or non-silencing control. Transfected cells were identified by fluorescence microscopy. Voltage response curves from patch clamp measurements are shown. Data points and lines representing the averages are shown. *, p < 0.05; **, p < 0.01 relative to controls.
Sc 391691 Pcp Biotin Jena Bioscience, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/plasmid+pcpg+free+lucia+pcpg+lucia/pm39862867-294-91-93?v=Jena+Bioscience
Average 95 stars, based on 1 article reviews
sc 391691 pcp biotin jena bioscience - by Bioz Stars, 2026-08
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paper pcs2 pcp 2xmcherry  (Addgene inc)
93
Addgene inc paper pcs2 pcp 2xmcherry
Bag1 regulates hERG expression. A, diagram of Bag1M transfection constructs used in this study. The UBL and BAG domain boundaries are marked with residue numbers, and the position of the R237A mutation disrupting <t>Hsp70</t> interaction is shown with a star. B, HeLa cells stably expressing hERG were transfected with siRNA against Bag1 (targeting all isoforms) or non-silencing control. CG and FG hERG were detected as 135- and 155-kDa bands, respectively, by immunoblot (IB) and quantified relative to the amount of each in control cells. Averages and standard deviations are shown to the right of data points. Knockdown of Bag1 was confirmed by immunoblot; the bands corresponding to endogenous Bag1L, Bag1M, and Bag1S are marked. C, experiment in B was performed with MG132 treatment, and hERG was quantified relative to MG132-treated non-silencing controls. D, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control. CG and FG hERG were detected and quantified as above. Bands corresponding to Bag1M, Bag1S, and C-Bag1 are marked. Lower panel, the experiment was performed with MG132 treatment and quantitation relative to MG132 treated vector control reported below the blot. E, HEK293 cells were transfected with human transferrin receptor (TfR) and Bag1 or vector control. The receptor was detected by immunoblot, and total amounts were quantified. F, HEK293 cells were transfected with hERG, GFP, and either Bag1 or vector control. Separately, HEK293 cells were transfected with hERG, siGLO, and either siRNA against Bag1 or non-silencing control. Transfected cells were identified by fluorescence microscopy. Voltage response curves from patch clamp measurements are shown. Data points and lines representing the averages are shown. *, p < 0.05; **, p < 0.01 relative to controls.
Paper Pcs2 Pcp 2xmcherry, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/plasmid+pcpg+free+lucia+pcpg+lucia/pmc10229589__41467_2023_38436_MOESM1_ESM-36-86-96?v=Addgene+inc
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pcp1 encoding gfp-vp22  (Becton Dickinson)
90
Becton Dickinson pcp1 encoding gfp-vp22
Bag1 regulates hERG expression. A, diagram of Bag1M transfection constructs used in this study. The UBL and BAG domain boundaries are marked with residue numbers, and the position of the R237A mutation disrupting <t>Hsp70</t> interaction is shown with a star. B, HeLa cells stably expressing hERG were transfected with siRNA against Bag1 (targeting all isoforms) or non-silencing control. CG and FG hERG were detected as 135- and 155-kDa bands, respectively, by immunoblot (IB) and quantified relative to the amount of each in control cells. Averages and standard deviations are shown to the right of data points. Knockdown of Bag1 was confirmed by immunoblot; the bands corresponding to endogenous Bag1L, Bag1M, and Bag1S are marked. C, experiment in B was performed with MG132 treatment, and hERG was quantified relative to MG132-treated non-silencing controls. D, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control. CG and FG hERG were detected and quantified as above. Bands corresponding to Bag1M, Bag1S, and C-Bag1 are marked. Lower panel, the experiment was performed with MG132 treatment and quantitation relative to MG132 treated vector control reported below the blot. E, HEK293 cells were transfected with human transferrin receptor (TfR) and Bag1 or vector control. The receptor was detected by immunoblot, and total amounts were quantified. F, HEK293 cells were transfected with hERG, GFP, and either Bag1 or vector control. Separately, HEK293 cells were transfected with hERG, siGLO, and either siRNA against Bag1 or non-silencing control. Transfected cells were identified by fluorescence microscopy. Voltage response curves from patch clamp measurements are shown. Data points and lines representing the averages are shown. *, p < 0.05; **, p < 0.01 relative to controls.
Pcp1 Encoding Gfp Vp22, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/plasmid+pcpg+free+lucia+pcpg+lucia/pmc01280190-57-1-40?v=Becton+Dickinson
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pcp1 encoding gfp-vp22 - by Bioz Stars, 2026-08
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cellular immunogen  (ImmunoGen Inc)
90
ImmunoGen Inc cellular immunogen
Bag1 regulates hERG expression. A, diagram of Bag1M transfection constructs used in this study. The UBL and BAG domain boundaries are marked with residue numbers, and the position of the R237A mutation disrupting <t>Hsp70</t> interaction is shown with a star. B, HeLa cells stably expressing hERG were transfected with siRNA against Bag1 (targeting all isoforms) or non-silencing control. CG and FG hERG were detected as 135- and 155-kDa bands, respectively, by immunoblot (IB) and quantified relative to the amount of each in control cells. Averages and standard deviations are shown to the right of data points. Knockdown of Bag1 was confirmed by immunoblot; the bands corresponding to endogenous Bag1L, Bag1M, and Bag1S are marked. C, experiment in B was performed with MG132 treatment, and hERG was quantified relative to MG132-treated non-silencing controls. D, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control. CG and FG hERG were detected and quantified as above. Bands corresponding to Bag1M, Bag1S, and C-Bag1 are marked. Lower panel, the experiment was performed with MG132 treatment and quantitation relative to MG132 treated vector control reported below the blot. E, HEK293 cells were transfected with human transferrin receptor (TfR) and Bag1 or vector control. The receptor was detected by immunoblot, and total amounts were quantified. F, HEK293 cells were transfected with hERG, GFP, and either Bag1 or vector control. Separately, HEK293 cells were transfected with hERG, siGLO, and either siRNA against Bag1 or non-silencing control. Transfected cells were identified by fluorescence microscopy. Voltage response curves from patch clamp measurements are shown. Data points and lines representing the averages are shown. *, p < 0.05; **, p < 0.01 relative to controls.
Cellular Immunogen, supplied by ImmunoGen Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/plasmid+pcpg+free+lucia+pcpg+lucia/us12018078-388-36-31?v=ImmunoGen+Inc
Average 90 stars, based on 1 article reviews
cellular immunogen - by Bioz Stars, 2026-08
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pcp7  (New England Biolabs)
97
New England Biolabs pcp7
Bag1 regulates hERG expression. A, diagram of Bag1M transfection constructs used in this study. The UBL and BAG domain boundaries are marked with residue numbers, and the position of the R237A mutation disrupting <t>Hsp70</t> interaction is shown with a star. B, HeLa cells stably expressing hERG were transfected with siRNA against Bag1 (targeting all isoforms) or non-silencing control. CG and FG hERG were detected as 135- and 155-kDa bands, respectively, by immunoblot (IB) and quantified relative to the amount of each in control cells. Averages and standard deviations are shown to the right of data points. Knockdown of Bag1 was confirmed by immunoblot; the bands corresponding to endogenous Bag1L, Bag1M, and Bag1S are marked. C, experiment in B was performed with MG132 treatment, and hERG was quantified relative to MG132-treated non-silencing controls. D, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control. CG and FG hERG were detected and quantified as above. Bands corresponding to Bag1M, Bag1S, and C-Bag1 are marked. Lower panel, the experiment was performed with MG132 treatment and quantitation relative to MG132 treated vector control reported below the blot. E, HEK293 cells were transfected with human transferrin receptor (TfR) and Bag1 or vector control. The receptor was detected by immunoblot, and total amounts were quantified. F, HEK293 cells were transfected with hERG, GFP, and either Bag1 or vector control. Separately, HEK293 cells were transfected with hERG, siGLO, and either siRNA against Bag1 or non-silencing control. Transfected cells were identified by fluorescence microscopy. Voltage response curves from patch clamp measurements are shown. Data points and lines representing the averages are shown. *, p < 0.05; **, p < 0.01 relative to controls.
Pcp7, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/plasmid+pcpg+free+lucia+pcpg+lucia/pm37695056-86-4-11?v=New+England+Biolabs
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Image Search Results


Bag1 regulates hERG expression. A, diagram of Bag1M transfection constructs used in this study. The UBL and BAG domain boundaries are marked with residue numbers, and the position of the R237A mutation disrupting Hsp70 interaction is shown with a star. B, HeLa cells stably expressing hERG were transfected with siRNA against Bag1 (targeting all isoforms) or non-silencing control. CG and FG hERG were detected as 135- and 155-kDa bands, respectively, by immunoblot (IB) and quantified relative to the amount of each in control cells. Averages and standard deviations are shown to the right of data points. Knockdown of Bag1 was confirmed by immunoblot; the bands corresponding to endogenous Bag1L, Bag1M, and Bag1S are marked. C, experiment in B was performed with MG132 treatment, and hERG was quantified relative to MG132-treated non-silencing controls. D, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control. CG and FG hERG were detected and quantified as above. Bands corresponding to Bag1M, Bag1S, and C-Bag1 are marked. Lower panel, the experiment was performed with MG132 treatment and quantitation relative to MG132 treated vector control reported below the blot. E, HEK293 cells were transfected with human transferrin receptor (TfR) and Bag1 or vector control. The receptor was detected by immunoblot, and total amounts were quantified. F, HEK293 cells were transfected with hERG, GFP, and either Bag1 or vector control. Separately, HEK293 cells were transfected with hERG, siGLO, and either siRNA against Bag1 or non-silencing control. Transfected cells were identified by fluorescence microscopy. Voltage response curves from patch clamp measurements are shown. Data points and lines representing the averages are shown. *, p < 0.05; **, p < 0.01 relative to controls.

Journal: The Journal of Biological Chemistry

Article Title: Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels *

doi: 10.1074/jbc.M116.752618

Figure Lengend Snippet: Bag1 regulates hERG expression. A, diagram of Bag1M transfection constructs used in this study. The UBL and BAG domain boundaries are marked with residue numbers, and the position of the R237A mutation disrupting Hsp70 interaction is shown with a star. B, HeLa cells stably expressing hERG were transfected with siRNA against Bag1 (targeting all isoforms) or non-silencing control. CG and FG hERG were detected as 135- and 155-kDa bands, respectively, by immunoblot (IB) and quantified relative to the amount of each in control cells. Averages and standard deviations are shown to the right of data points. Knockdown of Bag1 was confirmed by immunoblot; the bands corresponding to endogenous Bag1L, Bag1M, and Bag1S are marked. C, experiment in B was performed with MG132 treatment, and hERG was quantified relative to MG132-treated non-silencing controls. D, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control. CG and FG hERG were detected and quantified as above. Bands corresponding to Bag1M, Bag1S, and C-Bag1 are marked. Lower panel, the experiment was performed with MG132 treatment and quantitation relative to MG132 treated vector control reported below the blot. E, HEK293 cells were transfected with human transferrin receptor (TfR) and Bag1 or vector control. The receptor was detected by immunoblot, and total amounts were quantified. F, HEK293 cells were transfected with hERG, GFP, and either Bag1 or vector control. Separately, HEK293 cells were transfected with hERG, siGLO, and either siRNA against Bag1 or non-silencing control. Transfected cells were identified by fluorescence microscopy. Voltage response curves from patch clamp measurements are shown. Data points and lines representing the averages are shown. *, p < 0.05; **, p < 0.01 relative to controls.

Article Snippet: The following commercially available antibodies were used: Bag1 (Santa Cruz Biotechnology); CHIP (Abcam); goat anti-rabbit IgG-conjugated HRP (Jackson ImmunoResearch); goat anti-mouse IgG-conjugated HRP (Sigma); gp78 (Abcam); hemagglutinin (HA.11) (Covance); hERG (Alomone Labs); HRD1 (Abcam); Hsc70 (StressMarq); Hsp70 (StressMarq); Hsc/Hsp70 (StressGen); RMA1 (Abcam);TRC8 (Abcam); tubulin (Sigma); Ube2j1 (Abcam); Ube2g2 (Abcam); and ubiquitin (P4D1, Santa Cruz Biotechnology).

Techniques: Expressing, Transfection, Construct, Mutagenesis, Stable Transfection, Western Blot, Plasmid Preparation, Quantitation Assay, Fluorescence, Microscopy, Patch Clamp

Bag1 disrupts chaperone complexes with hERG. A, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control, and hERG was immunoprecipitated (IP). Co-precipitating Hsc70/Hsp70 and CHIP were detected by immunoblot (IB); Hsc70 and Hsp70 were equally detected by the antibody used here. Quantitation of Hsc70/Hsp70 and CHIP was adjusted for the total amount of immunoprecipitated hERG and plotted relative to the amounts in the control. Averages and standard deviations are shown to the right of data points. B, HeLa cells stably expressing hERG were transfected with siRNA against both Hsc70 and Hsp70, or against CHIP, or non-silencing control. CG and FG hERG were detected by immunoblot and quantified relative to the amount of each in control cells. C, cells as in B transfected with siRNA against Hsc70/Hsp70 were examined for hERG kinetics by pulse-chase as in Fig. 2A. Data points and lines representing the averages are shown. D, HEK293 cells were transfected with hERG and either Bag1 or vector control. Total light membrane fractions were isolated and treated with the indicated amounts of trypsin for 10 min at 37 °C. CG and FG hERG were detected by immunoblot and quantified relative to the amount without trypsin treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001 relative to controls.

Journal: The Journal of Biological Chemistry

Article Title: Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels *

doi: 10.1074/jbc.M116.752618

Figure Lengend Snippet: Bag1 disrupts chaperone complexes with hERG. A, HEK293 cells were transfected with hERG and the indicated Bag1 construct or vector control, and hERG was immunoprecipitated (IP). Co-precipitating Hsc70/Hsp70 and CHIP were detected by immunoblot (IB); Hsc70 and Hsp70 were equally detected by the antibody used here. Quantitation of Hsc70/Hsp70 and CHIP was adjusted for the total amount of immunoprecipitated hERG and plotted relative to the amounts in the control. Averages and standard deviations are shown to the right of data points. B, HeLa cells stably expressing hERG were transfected with siRNA against both Hsc70 and Hsp70, or against CHIP, or non-silencing control. CG and FG hERG were detected by immunoblot and quantified relative to the amount of each in control cells. C, cells as in B transfected with siRNA against Hsc70/Hsp70 were examined for hERG kinetics by pulse-chase as in Fig. 2A. Data points and lines representing the averages are shown. D, HEK293 cells were transfected with hERG and either Bag1 or vector control. Total light membrane fractions were isolated and treated with the indicated amounts of trypsin for 10 min at 37 °C. CG and FG hERG were detected by immunoblot and quantified relative to the amount without trypsin treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001 relative to controls.

Article Snippet: The following commercially available antibodies were used: Bag1 (Santa Cruz Biotechnology); CHIP (Abcam); goat anti-rabbit IgG-conjugated HRP (Jackson ImmunoResearch); goat anti-mouse IgG-conjugated HRP (Sigma); gp78 (Abcam); hemagglutinin (HA.11) (Covance); hERG (Alomone Labs); HRD1 (Abcam); Hsc70 (StressMarq); Hsp70 (StressMarq); Hsc/Hsp70 (StressGen); RMA1 (Abcam);TRC8 (Abcam); tubulin (Sigma); Ube2j1 (Abcam); Ube2g2 (Abcam); and ubiquitin (P4D1, Santa Cruz Biotechnology).

Techniques: Transfection, Construct, Plasmid Preparation, Immunoprecipitation, Western Blot, Quantitation Assay, Stable Transfection, Expressing, Pulse Chase, Isolation

TRC8 decreases trafficking and function of hERG. A, HEK293 cells were transfected with hERG and the indicated TRC8 construct or vector control and examined for hERG kinetics by pulse-chase as in Fig. 2A. Data points and lines representing the averages are shown. B, HEK293 cells were transfected with hERG, GFP, and either TRC8 or vector control. Voltage-response curves from patch clamp measurements as in Fig. 1E are shown. C, cells were transfected as in A with the indicated HA-tagged TRC8 construct, and hERG was immunoprecipitated (IP). Co-precipitating TRC8 was detected by immunoblot using specific antibodies against TRC8 and the HA tag, and Hsp70 was detected as a control. Quantitation of TRC8 was adjusted for the total amount of immunoprecipitated hERG and plotted relative to the amounts in the control. *, p < 0.05; **, p < 0.01 relative to controls.

Journal: The Journal of Biological Chemistry

Article Title: Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels *

doi: 10.1074/jbc.M116.752618

Figure Lengend Snippet: TRC8 decreases trafficking and function of hERG. A, HEK293 cells were transfected with hERG and the indicated TRC8 construct or vector control and examined for hERG kinetics by pulse-chase as in Fig. 2A. Data points and lines representing the averages are shown. B, HEK293 cells were transfected with hERG, GFP, and either TRC8 or vector control. Voltage-response curves from patch clamp measurements as in Fig. 1E are shown. C, cells were transfected as in A with the indicated HA-tagged TRC8 construct, and hERG was immunoprecipitated (IP). Co-precipitating TRC8 was detected by immunoblot using specific antibodies against TRC8 and the HA tag, and Hsp70 was detected as a control. Quantitation of TRC8 was adjusted for the total amount of immunoprecipitated hERG and plotted relative to the amounts in the control. *, p < 0.05; **, p < 0.01 relative to controls.

Article Snippet: The following commercially available antibodies were used: Bag1 (Santa Cruz Biotechnology); CHIP (Abcam); goat anti-rabbit IgG-conjugated HRP (Jackson ImmunoResearch); goat anti-mouse IgG-conjugated HRP (Sigma); gp78 (Abcam); hemagglutinin (HA.11) (Covance); hERG (Alomone Labs); HRD1 (Abcam); Hsc70 (StressMarq); Hsp70 (StressMarq); Hsc/Hsp70 (StressGen); RMA1 (Abcam);TRC8 (Abcam); tubulin (Sigma); Ube2j1 (Abcam); Ube2g2 (Abcam); and ubiquitin (P4D1, Santa Cruz Biotechnology).

Techniques: Transfection, Construct, Plasmid Preparation, Pulse Chase, Patch Clamp, Immunoprecipitation, Western Blot, Quantitation Assay