sar1a  (OriGene)

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Mutant SAR1A and SAR1B GTPases with dominant negative mutations T39N and H79G decrease the cell surface expression of Nav1.5. (A) Western blot analysis of Nav1.5 using plasma membrane protein extracts or total cellular lysates from HEK293 cells transfected with expression plasmids for wild type (WT) SAR1A-Flag, SAR1A:T39N-Flag, and SAR1A:H79G-Flag, or the empty vector control. The plasma membrane protein extracts were prepared using the biotinylation procedure. (B) Quantified data from Western blot analysis as in (A) showing the relative amount of Nav1.5 over tubulin in total cell lysates. (C) Quantified data from Western blot analysis as in (A) showing the relative amount of Nav1.5 over tubulin in plasma membranes over Nav1.5 over tubulin in total cell lysates. (D) Western blot analysis of Nav1.5 using plasma membrane proteins extracts or total cellular lysates from HEK293 cells transfected with expression plasmids for wild type SAR1B-Flag, SAR1B:T39N-Flag, and SAR1B:H79G-Flag, or the empty vector control. The plasma membrane proteins extracts were prepared using the biotinylation procedure. (E) Quantified data from Western blot analysis as in (D) showing the relative amount of Nav1.5 over tubulin in total cell lysates. (F) Quantified data from Western blot analysis as in (D) showing the relative amount of Nav1.5 in plasma membranes over Nav1.5 in total cell lysates. All studies were repeated at least three times. Data are shown as mean ± SEM. NS, not significant; *P < 0.05 (n=3).

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Mutagenesis, Dominant Negative Mutation, Expressing, Western Blot, Clinical Proteomics, Membrane, Transfection, Plasmid Preparation, Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Mutant SAR1A GTPases with dominant negative mutations T39N and H79G decrease sodium current INa in HEK/Nav1.5 cells. (A) Representative whole-cell sodium current traces recorded from HEK/Nav1.5 cells transfected with empty vector control or expression plasmids for wild type (WT) SAR1A-Flag, SAR1A:T39N-Flag or SAR1A:H79G-Flag. (B) The relationship between the average current density (current normalized to cell capacitance) and voltage. The voltage clamp protocol was shown in the inset. (C) The relative peak sodium current density at −25 mV among different treatment groups. Current density (pA/pF) was - 192.16±19.70 (n=15 cells) for the empty vector control, −50.05±12.52 (n=9 cells) for T39N-SAR1A-FLAG, −68.39±16.87 (n=12 cells) for H79G-SAR1A-FLAG, or −189.03±34.21 (n=13 cells) for WT-SAR1A-FLAG. Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Mutagenesis, Dominant Negative Mutation, Transfection, Plasmid Preparation, Control, Expressing

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Mutant SAR1A and SAR1B GTPases with dominant negative mutations T39N and H79G decrease INa in neonatal rat cardiomyocytes. (A) Representative whole-cell sodium current traces recorded from neonatal rat cardiomyocytes transfected with the empty vector control or expression plasmids for wild type (WT) SAR1A-Flag, SAR1A:H79G-Flag, WT-SAR1B-Flag. SAR1B:T39N-Flag or SAR1B:H79G-Flag, respectively. (B) The relationship between the average current density (current normalized to cell capacitance) and voltage. The voltage clamp protocol was shown in the inset. (C) The relative peak sodium current density at −30 mV among different treatment groups. Current densities (pA/pF) were −538.63±55.99 (n=26 cells) for the empty vector control, −276.64±36.80 (n=25 cells) for T39N-SAR1A-Flag, −265.22±27.91 (n=29 cells) for H79G-SAR1A-Flag, −552.17±51.53 (n=26 cells) for WT-SAR1A-Flag, −216.39±28.29 (n=28 cells) for SAR1B:T39N-Flag, −288.51±34.55 (n=30 cells) for SAR1B:H79G-Flag, and −570.23 ± 49.88 (n=26 cells) for WT-SAR1B-Flag. Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Mutagenesis, Dominant Negative Mutation, Transfection, Plasmid Preparation, Control, Expressing

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Knockdown of either SAR1A or SAR1B expression does not affect INa in HEK/Nav1.5 cells. (A, E) Real-time RT-PCR analysis showing successful knockdown of SAR1A or SAR1B expression in HEK/Nav1.5 cells transfected with siRNA to SAR1A or SAR1B and scrambler siRNA (NC) as a control. (B, F) Representative whole-cell sodium current traces recorded from HEK/Nav1.5 cells transfected with SAR1A and SAR1B siRNAs or NC siRNAs. (C, G) The relationship of average current densities (current normalized to cell capacitance) and voltage in HEK/Nav1.5 cells transfected with SAR1A siRNAs or SAR1B siRNA compared to NC siRNA. The voltage clamp protocol was shown in the inset. (D) Relative peak sodium current densities (pA/pF) for SAR1A siRNAs at −25 mV. Current densities (pA/pF) were −137.69±25.35 for SAR1A siRNA1 (n=11 cells), −112.22+18.12 for SAR1A siRNA2 (n=12 cells) and −168.35±29.55 for NC siRNA (11 cells). (H) Relative peak sodium current densities (pA/pF) for SAR1B siRNA at −25 mV. Current densities (pA/pF) were −156.82±16.97 for SAR1B siRNA1 (n=11 cells), and −157.70±24.03 for NC siRNA (12 cells). Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Transfection, Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Simultaneous double knockdown of SAR1A and SAR1B expression by siRNAs significantly reduces INa density in HEK/Nav1.5 cells. (A) Real-time RT-PCR analysis showing successful knockdown of SAR1A expression in HEK/Nav1.5 cells transfected with SAR1A siRNA1+SAR1B siRNA1 or SAR1A siRNA2+SAR1B siRNA1 as compared with scrambler negative control siRNA (NC). (B) Real-time RT-PCR analysis showing successful knockdown of SAR1B expression in HEK/Nav1.5 cells transfected with SAR1A siRNA1+SAR1B siRNA1 or SAR1A siRNA2+SAR1B siRNA1 as compared with scrambler negative control siRNA (NC). (C) Representative whole-cell sodium current traces. (D) The relationship of average current densities (current normalized to cell capacitance) and voltage. The voltage clamp protocol was shown in the inset. (E) Relative peak sodium current densities (pA/pF) at −25 mV. Current densities (pA/pF) were −128.97±12.56 for SAR1A siRNA1+SAR1B siRNA1 (n=24), −71.31+10.94 for SAR1A siRNA2+SAR1B siRNA1 (n=23) and −189.25±21.57 for NC siRNA (n=24). Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Transfection, Negative Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: SAR1A and SAR1B interact with MOG1. (A) GST-pulldown analysis showed that Flag-MOG1 overexpressed in HEK293 cells was pulled down by either GST-SAR1A or GST-SAR1B, but not by GST. (B) Co-IP analysis showed that Flag-MOG1 in HEK293 cells was successfully precipitated by an anti-GFP antibody recognizing GFP-SAR1A, but bot by the control anti-mouse IgG. (C) Co-IP analysis showed that Flag-MOG1 in HEK293 cells was successfully precipitated by an anti-GFP antibody recognizing GFP-SAR1B, but bot by the control anti-mouse IgG. (D) Reciprocal Co-IP analysis showed that GFP-SAR1A in HEK293 cells was successfully precipitated by an anti-FLAG antibody recognizing Flag-MOG1, but not by the control anti-rabbit IgG. (E) Reciprocal Co-IP analysis showed that GFP-SAR1B in HEK293 cells was successfully precipitated by an anti-FLAG antibody recognizing Flag-MOG1, but not by the control anti-rabbit IgG. (F) GST-pulldown analysis showed that Flag-MOG1 in HEK293 cells was successfully pulled down by GST-SAR1A:T39N, GST-SAR1A:H79G, GST-SAR1B:T39N or GST-SAR1B:H79G, but not GST alone. (G) Top image: Co-IP analysis showed that the anti-Flag antibody recognizing Flag-MOG1, but not the control anti-rabbit IgG, successfully precipitated GFP-SAR1A, GFP-SAR1A:T39N or GFP-SAR1A:H79G. Bottom image: Reciprocal Co-IP analysis showed that the anti-GFP antibody recognizing GFP-SAR1A, GFP-SAR1A:T39N or GFP-SAR1A:H79G, but not the anti-mouse IgG, successfully precipitated Flag-MOG1. (H) Top image: Co-IP analysis showed that the anti-Flag antibody recognizing Flag-MOG1, but not the control anti-rabbit IgG, successfully precipitated GFP-SAR1B, GFP-SAR1B:T39N or GFP-SAR1B:H79G. Bottom image: Reciprocal Co-IP analysis showed that anti-GFP antibody recognizing GFP-SAR1B, GFP-SAR1B:T39N or GFP-SAR1B:H79G, but not the control anti-rabbit IgG, successfully precipitated Flag-MOG1.

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: MOG1-increased INa density is abolished when SAR1A and SAR1B expression is knocked down. (A) Representative whole-cell sodium current traces recorded from HEK/Nav1.5 cells transfected with the empty vector control vs. a MOG1 expression plasmid in combination with SAR1AB siRNAs (SAR1A siRNA2 + SAR1B siRNA1) vs. negative control (NC) siRNA. (B) Relationship of average current densities (current normalized to cell capacitance) and voltage. The voltage clamp protocol was shown in the inset. (C) Relative peak sodium current densities (pA/pF) at −25 mV. Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Negative Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: MOG1-mediated plasma membrane trafficking of Nav1.5 is abolished when SAR1A and SAR1B expression is knocked down. (A) Western blot analysis of Nav1.5 using either biotinylated plasma membrane protein extracts or total cellular lysates from HEK293 cells transfected with the empty vector control vs. a MOG1 expression plasmid in combination with SAR1A/B siRNAs vs. negative control (NC) siRNA. Tubulin was used as a loading control. (B) Quantified data from Western blot analysis as in (A) showing the relative amount of Nav1.5 over tubulin in total cellular lysates. (C) Quantified data from Western blot analysis as in (A) showing the relative amount of Nav1.5 over tubulin in plasma membranes over Nav1.5 over tubulin in total cellular lysates. All studies were repeated at least three times. Data are shown as mean ± SEM. NS, not significant; *P < 0.05 (n=3).

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Clinical Proteomics, Membrane, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Negative Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Mutant SAR1A and SAR1B GTPases with dominant negative mutations T39N and H79G decrease the cell surface expression of Nav1.5. (A) Western blot analysis of Nav1.5 using plasma membrane protein extracts or total cellular lysates from HEK293 cells transfected with expression plasmids for wild type (WT) SAR1A-Flag, SAR1A:T39N-Flag, and SAR1A:H79G-Flag, or the empty vector control. The plasma membrane protein extracts were prepared using the biotinylation procedure. (B) Quantified data from Western blot analysis as in (A) showing the relative amount of Nav1.5 over tubulin in total cell lysates. (C) Quantified data from Western blot analysis as in (A) showing the relative amount of Nav1.5 over tubulin in plasma membranes over Nav1.5 over tubulin in total cell lysates. (D) Western blot analysis of Nav1.5 using plasma membrane proteins extracts or total cellular lysates from HEK293 cells transfected with expression plasmids for wild type SAR1B-Flag, SAR1B:T39N-Flag, and SAR1B:H79G-Flag, or the empty vector control. The plasma membrane proteins extracts were prepared using the biotinylation procedure. (E) Quantified data from Western blot analysis as in (D) showing the relative amount of Nav1.5 over tubulin in total cell lysates. (F) Quantified data from Western blot analysis as in (D) showing the relative amount of Nav1.5 in plasma membranes over Nav1.5 in total cell lysates. All studies were repeated at least three times. Data are shown as mean ± SEM. NS, not significant; *P < 0.05 (n=3).

Article Snippet: RNA interference The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Mutagenesis, Dominant Negative Mutation, Expressing, Western Blot, Clinical Proteomics, Membrane, Transfection, Plasmid Preparation, Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Mutant SAR1B GTPases with dominant negative mutations T39N and H79G decrease densities of sodium current INa in HEK/Nav1.5 cells. (A) Representative whole-cell sodium current traces recorded from HEK/Nav1.5 cells transfected with the empty vector control or expression plasmids for wild type SAR1B-Flag, SAR1B:T39N-Flag or SAR1B:H79G-Flag. (B) The relationship between the average current density (current normalized to cell capacitance) and voltage. The voltage clamp protocol was shown in the inset. (C) The relative peak sodium current density at −25 mV among different treatment groups. Current densities (pA/pF) were −145.57±28.87 (n=12 cells) for empty vector control, −32.55±3.76 (n=13 cells) for SAR1B:T39N-FLAG, −42.97±17.19 (n=9 cells) for SAR1B:H79G-FLAG, and - 139.08±29.98 (n=9 cells) for WT-SAR1B-FLAG. Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: RNA interference The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Mutagenesis, Dominant Negative Mutation, Transfection, Plasmid Preparation, Control, Expressing

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Mutant SAR1A and SAR1B GTPases with dominant negative mutations T39N and H79G decrease INa in neonatal rat cardiomyocytes. (A) Representative whole-cell sodium current traces recorded from neonatal rat cardiomyocytes transfected with the empty vector control or expression plasmids for wild type (WT) SAR1A-Flag, SAR1A:H79G-Flag, WT-SAR1B-Flag. SAR1B:T39N-Flag or SAR1B:H79G-Flag, respectively. (B) The relationship between the average current density (current normalized to cell capacitance) and voltage. The voltage clamp protocol was shown in the inset. (C) The relative peak sodium current density at −30 mV among different treatment groups. Current densities (pA/pF) were −538.63±55.99 (n=26 cells) for the empty vector control, −276.64±36.80 (n=25 cells) for T39N-SAR1A-Flag, −265.22±27.91 (n=29 cells) for H79G-SAR1A-Flag, −552.17±51.53 (n=26 cells) for WT-SAR1A-Flag, −216.39±28.29 (n=28 cells) for SAR1B:T39N-Flag, −288.51±34.55 (n=30 cells) for SAR1B:H79G-Flag, and −570.23 ± 49.88 (n=26 cells) for WT-SAR1B-Flag. Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: RNA interference The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Mutagenesis, Dominant Negative Mutation, Transfection, Plasmid Preparation, Control, Expressing

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Knockdown of either SAR1A or SAR1B expression does not affect INa in HEK/Nav1.5 cells. (A, E) Real-time RT-PCR analysis showing successful knockdown of SAR1A or SAR1B expression in HEK/Nav1.5 cells transfected with siRNA to SAR1A or SAR1B and scrambler siRNA (NC) as a control. (B, F) Representative whole-cell sodium current traces recorded from HEK/Nav1.5 cells transfected with SAR1A and SAR1B siRNAs or NC siRNAs. (C, G) The relationship of average current densities (current normalized to cell capacitance) and voltage in HEK/Nav1.5 cells transfected with SAR1A siRNAs or SAR1B siRNA compared to NC siRNA. The voltage clamp protocol was shown in the inset. (D) Relative peak sodium current densities (pA/pF) for SAR1A siRNAs at −25 mV. Current densities (pA/pF) were −137.69±25.35 for SAR1A siRNA1 (n=11 cells), −112.22+18.12 for SAR1A siRNA2 (n=12 cells) and −168.35±29.55 for NC siRNA (11 cells). (H) Relative peak sodium current densities (pA/pF) for SAR1B siRNA at −25 mV. Current densities (pA/pF) were −156.82±16.97 for SAR1B siRNA1 (n=11 cells), and −157.70±24.03 for NC siRNA (12 cells). Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: RNA interference The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Transfection, Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Simultaneous double knockdown of SAR1A and SAR1B expression by siRNAs significantly reduces INa density in HEK/Nav1.5 cells. (A) Real-time RT-PCR analysis showing successful knockdown of SAR1A expression in HEK/Nav1.5 cells transfected with SAR1A siRNA1+SAR1B siRNA1 or SAR1A siRNA2+SAR1B siRNA1 as compared with scrambler negative control siRNA (NC). (B) Real-time RT-PCR analysis showing successful knockdown of SAR1B expression in HEK/Nav1.5 cells transfected with SAR1A siRNA1+SAR1B siRNA1 or SAR1A siRNA2+SAR1B siRNA1 as compared with scrambler negative control siRNA (NC). (C) Representative whole-cell sodium current traces. (D) The relationship of average current densities (current normalized to cell capacitance) and voltage. The voltage clamp protocol was shown in the inset. (E) Relative peak sodium current densities (pA/pF) at −25 mV. Current densities (pA/pF) were −128.97±12.56 for SAR1A siRNA1+SAR1B siRNA1 (n=24), −71.31+10.94 for SAR1A siRNA2+SAR1B siRNA1 (n=23) and −189.25±21.57 for NC siRNA (n=24). Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: RNA interference The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Transfection, Negative Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: SAR1A and SAR1B interact with MOG1. (A) GST-pulldown analysis showed that Flag-MOG1 overexpressed in HEK293 cells was pulled down by either GST-SAR1A or GST-SAR1B, but not by GST. (B) Co-IP analysis showed that Flag-MOG1 in HEK293 cells was successfully precipitated by an anti-GFP antibody recognizing GFP-SAR1A, but bot by the control anti-mouse IgG. (C) Co-IP analysis showed that Flag-MOG1 in HEK293 cells was successfully precipitated by an anti-GFP antibody recognizing GFP-SAR1B, but bot by the control anti-mouse IgG. (D) Reciprocal Co-IP analysis showed that GFP-SAR1A in HEK293 cells was successfully precipitated by an anti-FLAG antibody recognizing Flag-MOG1, but not by the control anti-rabbit IgG. (E) Reciprocal Co-IP analysis showed that GFP-SAR1B in HEK293 cells was successfully precipitated by an anti-FLAG antibody recognizing Flag-MOG1, but not by the control anti-rabbit IgG. (F) GST-pulldown analysis showed that Flag-MOG1 in HEK293 cells was successfully pulled down by GST-SAR1A:T39N, GST-SAR1A:H79G, GST-SAR1B:T39N or GST-SAR1B:H79G, but not GST alone. (G) Top image: Co-IP analysis showed that the anti-Flag antibody recognizing Flag-MOG1, but not the control anti-rabbit IgG, successfully precipitated GFP-SAR1A, GFP-SAR1A:T39N or GFP-SAR1A:H79G. Bottom image: Reciprocal Co-IP analysis showed that the anti-GFP antibody recognizing GFP-SAR1A, GFP-SAR1A:T39N or GFP-SAR1A:H79G, but not the anti-mouse IgG, successfully precipitated Flag-MOG1. (H) Top image: Co-IP analysis showed that the anti-Flag antibody recognizing Flag-MOG1, but not the control anti-rabbit IgG, successfully precipitated GFP-SAR1B, GFP-SAR1B:T39N or GFP-SAR1B:H79G. Bottom image: Reciprocal Co-IP analysis showed that anti-GFP antibody recognizing GFP-SAR1B, GFP-SAR1B:T39N or GFP-SAR1B:H79G, but not the control anti-rabbit IgG, successfully precipitated Flag-MOG1.

Article Snippet: RNA interference The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: MOG1-increased INa density is abolished when SAR1A and SAR1B expression is knocked down. (A) Representative whole-cell sodium current traces recorded from HEK/Nav1.5 cells transfected with the empty vector control vs. a MOG1 expression plasmid in combination with SAR1AB siRNAs (SAR1A siRNA2 + SAR1B siRNA1) vs. negative control (NC) siRNA. (B) Relationship of average current densities (current normalized to cell capacitance) and voltage. The voltage clamp protocol was shown in the inset. (C) Relative peak sodium current densities (pA/pF) at −25 mV. Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: RNA interference The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Negative Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: MOG1-mediated plasma membrane trafficking of Nav1.5 is abolished when SAR1A and SAR1B expression is knocked down. (A) Western blot analysis of Nav1.5 using either biotinylated plasma membrane protein extracts or total cellular lysates from HEK293 cells transfected with the empty vector control vs. a MOG1 expression plasmid in combination with SAR1A/B siRNAs vs. negative control (NC) siRNA. Tubulin was used as a loading control. (B) Quantified data from Western blot analysis as in (A) showing the relative amount of Nav1.5 over tubulin in total cellular lysates. (C) Quantified data from Western blot analysis as in (A) showing the relative amount of Nav1.5 over tubulin in plasma membranes over Nav1.5 over tubulin in total cellular lysates. All studies were repeated at least three times. Data are shown as mean ± SEM. NS, not significant; *P < 0.05 (n=3).

Article Snippet: RNA interference The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Clinical Proteomics, Membrane, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Negative Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Mutant SAR1A and SAR1B GTPases with dominant negative mutations T39N and H79G decrease the cell surface expression of Nav1.5. (A) Western blot analysis of Nav1.5 using plasma membrane protein extracts or total cellular lysates from HEK293 cells transfected with expression plasmids for wild type (WT) SAR1A-Flag, SAR1A:T39N-Flag, and SAR1A:H79G-Flag, or the empty vector control. The plasma membrane protein extracts were prepared using the biotinylation procedure. (B) Quantified data from Western blot analysis as in (A) showing the relative amount of Nav1.5 over tubulin in total cell lysates. (C) Quantified data from Western blot analysis as in (A) showing the relative amount of Nav1.5 over tubulin in plasma membranes over Nav1.5 over tubulin in total cell lysates. (D) Western blot analysis of Nav1.5 using plasma membrane proteins extracts or total cellular lysates from HEK293 cells transfected with expression plasmids for wild type SAR1B-Flag, SAR1B:T39N-Flag, and SAR1B:H79G-Flag, or the empty vector control. The plasma membrane proteins extracts were prepared using the biotinylation procedure. (E) Quantified data from Western blot analysis as in (D) showing the relative amount of Nav1.5 over tubulin in total cell lysates. (F) Quantified data from Western blot analysis as in (D) showing the relative amount of Nav1.5 in plasma membranes over Nav1.5 in total cell lysates. All studies were repeated at least three times. Data are shown as mean ± SEM. NS, not significant; *P < 0.05 (n=3).

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Mutagenesis, Dominant Negative Mutation, Expressing, Western Blot, Clinical Proteomics, Membrane, Transfection, Plasmid Preparation, Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Mutant SAR1B GTPases with dominant negative mutations T39N and H79G decrease densities of sodium current INa in HEK/Nav1.5 cells. (A) Representative whole-cell sodium current traces recorded from HEK/Nav1.5 cells transfected with the empty vector control or expression plasmids for wild type SAR1B-Flag, SAR1B:T39N-Flag or SAR1B:H79G-Flag. (B) The relationship between the average current density (current normalized to cell capacitance) and voltage. The voltage clamp protocol was shown in the inset. (C) The relative peak sodium current density at −25 mV among different treatment groups. Current densities (pA/pF) were −145.57±28.87 (n=12 cells) for empty vector control, −32.55±3.76 (n=13 cells) for SAR1B:T39N-FLAG, −42.97±17.19 (n=9 cells) for SAR1B:H79G-FLAG, and - 139.08±29.98 (n=9 cells) for WT-SAR1B-FLAG. Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Mutagenesis, Dominant Negative Mutation, Transfection, Plasmid Preparation, Control, Expressing

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Mutant SAR1A and SAR1B GTPases with dominant negative mutations T39N and H79G decrease INa in neonatal rat cardiomyocytes. (A) Representative whole-cell sodium current traces recorded from neonatal rat cardiomyocytes transfected with the empty vector control or expression plasmids for wild type (WT) SAR1A-Flag, SAR1A:H79G-Flag, WT-SAR1B-Flag. SAR1B:T39N-Flag or SAR1B:H79G-Flag, respectively. (B) The relationship between the average current density (current normalized to cell capacitance) and voltage. The voltage clamp protocol was shown in the inset. (C) The relative peak sodium current density at −30 mV among different treatment groups. Current densities (pA/pF) were −538.63±55.99 (n=26 cells) for the empty vector control, −276.64±36.80 (n=25 cells) for T39N-SAR1A-Flag, −265.22±27.91 (n=29 cells) for H79G-SAR1A-Flag, −552.17±51.53 (n=26 cells) for WT-SAR1A-Flag, −216.39±28.29 (n=28 cells) for SAR1B:T39N-Flag, −288.51±34.55 (n=30 cells) for SAR1B:H79G-Flag, and −570.23 ± 49.88 (n=26 cells) for WT-SAR1B-Flag. Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Mutagenesis, Dominant Negative Mutation, Transfection, Plasmid Preparation, Control, Expressing

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Knockdown of either SAR1A or SAR1B expression does not affect INa in HEK/Nav1.5 cells. (A, E) Real-time RT-PCR analysis showing successful knockdown of SAR1A or SAR1B expression in HEK/Nav1.5 cells transfected with siRNA to SAR1A or SAR1B and scrambler siRNA (NC) as a control. (B, F) Representative whole-cell sodium current traces recorded from HEK/Nav1.5 cells transfected with SAR1A and SAR1B siRNAs or NC siRNAs. (C, G) The relationship of average current densities (current normalized to cell capacitance) and voltage in HEK/Nav1.5 cells transfected with SAR1A siRNAs or SAR1B siRNA compared to NC siRNA. The voltage clamp protocol was shown in the inset. (D) Relative peak sodium current densities (pA/pF) for SAR1A siRNAs at −25 mV. Current densities (pA/pF) were −137.69±25.35 for SAR1A siRNA1 (n=11 cells), −112.22+18.12 for SAR1A siRNA2 (n=12 cells) and −168.35±29.55 for NC siRNA (11 cells). (H) Relative peak sodium current densities (pA/pF) for SAR1B siRNA at −25 mV. Current densities (pA/pF) were −156.82±16.97 for SAR1B siRNA1 (n=11 cells), and −157.70±24.03 for NC siRNA (12 cells). Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Transfection, Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: Simultaneous double knockdown of SAR1A and SAR1B expression by siRNAs significantly reduces INa density in HEK/Nav1.5 cells. (A) Real-time RT-PCR analysis showing successful knockdown of SAR1A expression in HEK/Nav1.5 cells transfected with SAR1A siRNA1+SAR1B siRNA1 or SAR1A siRNA2+SAR1B siRNA1 as compared with scrambler negative control siRNA (NC). (B) Real-time RT-PCR analysis showing successful knockdown of SAR1B expression in HEK/Nav1.5 cells transfected with SAR1A siRNA1+SAR1B siRNA1 or SAR1A siRNA2+SAR1B siRNA1 as compared with scrambler negative control siRNA (NC). (C) Representative whole-cell sodium current traces. (D) The relationship of average current densities (current normalized to cell capacitance) and voltage. The voltage clamp protocol was shown in the inset. (E) Relative peak sodium current densities (pA/pF) at −25 mV. Current densities (pA/pF) were −128.97±12.56 for SAR1A siRNA1+SAR1B siRNA1 (n=24), −71.31+10.94 for SAR1A siRNA2+SAR1B siRNA1 (n=23) and −189.25±21.57 for NC siRNA (n=24). Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Transfection, Negative Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: SAR1A and SAR1B interact with MOG1. (A) GST-pulldown analysis showed that Flag-MOG1 overexpressed in HEK293 cells was pulled down by either GST-SAR1A or GST-SAR1B, but not by GST. (B) Co-IP analysis showed that Flag-MOG1 in HEK293 cells was successfully precipitated by an anti-GFP antibody recognizing GFP-SAR1A, but bot by the control anti-mouse IgG. (C) Co-IP analysis showed that Flag-MOG1 in HEK293 cells was successfully precipitated by an anti-GFP antibody recognizing GFP-SAR1B, but bot by the control anti-mouse IgG. (D) Reciprocal Co-IP analysis showed that GFP-SAR1A in HEK293 cells was successfully precipitated by an anti-FLAG antibody recognizing Flag-MOG1, but not by the control anti-rabbit IgG. (E) Reciprocal Co-IP analysis showed that GFP-SAR1B in HEK293 cells was successfully precipitated by an anti-FLAG antibody recognizing Flag-MOG1, but not by the control anti-rabbit IgG. (F) GST-pulldown analysis showed that Flag-MOG1 in HEK293 cells was successfully pulled down by GST-SAR1A:T39N, GST-SAR1A:H79G, GST-SAR1B:T39N or GST-SAR1B:H79G, but not GST alone. (G) Top image: Co-IP analysis showed that the anti-Flag antibody recognizing Flag-MOG1, but not the control anti-rabbit IgG, successfully precipitated GFP-SAR1A, GFP-SAR1A:T39N or GFP-SAR1A:H79G. Bottom image: Reciprocal Co-IP analysis showed that the anti-GFP antibody recognizing GFP-SAR1A, GFP-SAR1A:T39N or GFP-SAR1A:H79G, but not the anti-mouse IgG, successfully precipitated Flag-MOG1. (H) Top image: Co-IP analysis showed that the anti-Flag antibody recognizing Flag-MOG1, but not the control anti-rabbit IgG, successfully precipitated GFP-SAR1B, GFP-SAR1B:T39N or GFP-SAR1B:H79G. Bottom image: Reciprocal Co-IP analysis showed that anti-GFP antibody recognizing GFP-SAR1B, GFP-SAR1B:T39N or GFP-SAR1B:H79G, but not the control anti-rabbit IgG, successfully precipitated Flag-MOG1.

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: MOG1-increased INa density is abolished when SAR1A and SAR1B expression is knocked down. (A) Representative whole-cell sodium current traces recorded from HEK/Nav1.5 cells transfected with the empty vector control vs. a MOG1 expression plasmid in combination with SAR1AB siRNAs (SAR1A siRNA2 + SAR1B siRNA1) vs. negative control (NC) siRNA. (B) Relationship of average current densities (current normalized to cell capacitance) and voltage. The voltage clamp protocol was shown in the inset. (C) Relative peak sodium current densities (pA/pF) at −25 mV. Data are shown as mean ± SEM. NS, not significant; *P < 0.05.

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Negative Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Na v 1.5

doi: 10.1016/j.bbadis.2018.09.003

Figure Lengend Snippet: MOG1-mediated plasma membrane trafficking of Nav1.5 is abolished when SAR1A and SAR1B expression is knocked down. (A) Western blot analysis of Nav1.5 using either biotinylated plasma membrane protein extracts or total cellular lysates from HEK293 cells transfected with the empty vector control vs. a MOG1 expression plasmid in combination with SAR1A/B siRNAs vs. negative control (NC) siRNA. Tubulin was used as a loading control. (B) Quantified data from Western blot analysis as in (A) showing the relative amount of Nav1.5 over tubulin in total cellular lysates. (C) Quantified data from Western blot analysis as in (A) showing the relative amount of Nav1.5 over tubulin in plasma membranes over Nav1.5 over tubulin in total cellular lysates. All studies were repeated at least three times. Data are shown as mean ± SEM. NS, not significant; *P < 0.05 (n=3).

Article Snippet: The siRNAs against human SAR1A , SAR1B and scrambled control siRNA were designed and synthesized by GenePharma (Suzhou, Jiangsu, China).

Techniques: Clinical Proteomics, Membrane, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Negative Control