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    Syntaxin αsnap
    Figure 1. siRNA-mediated downregulation of <t>αSNAP</t> alters the autophagic flux. ( A ) SK-CO15 cells were transfected with either control or two different αSNAP-specific siRNA duplexes (D1 and D2). Expression of αSNAP and autophagic markers, LC3, NBR1 and p62 in total-cell lysates was determined by immunoblotting. ( B ) siRNA depletion of αSNAP was performed in either control SK-CO15 cells (SK-neo) or cells with stable expression of siRNA-resistant bovine αSNAP (SK-αSNAP). Expression of αSNAP and autophagic markers in cell lysates was determined by immunoblotting 48 h post-transfection. ( C and D ) HeLa-GFP-LC3 cells were transfected with either control or αSNAP-specific siRNAs and formation of autophagosomes was visualized by confocal microscopy analysis of GFP fluorescence in fixed cells 72 h post-transfection. Data in this and other figures are presented as mean ± SEM of three independent experiments. *p
    αsnap, supplied by Syntaxin, used in various techniques. Bioz Stars score: 90/100, based on 270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/αsnap/product/Syntaxin
    Average 90 stars, based on 270 article reviews
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    αsnap - by Bioz Stars, 2020-08
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    86
    Abcam anti αsnap
    Figure 1. siRNA-mediated downregulation of <t>αSNAP</t> alters the autophagic flux. ( A ) SK-CO15 cells were transfected with either control or two different αSNAP-specific siRNA duplexes (D1 and D2). Expression of αSNAP and autophagic markers, LC3, NBR1 and p62 in total-cell lysates was determined by immunoblotting. ( B ) siRNA depletion of αSNAP was performed in either control SK-CO15 cells (SK-neo) or cells with stable expression of siRNA-resistant bovine αSNAP (SK-αSNAP). Expression of αSNAP and autophagic markers in cell lysates was determined by immunoblotting 48 h post-transfection. ( C and D ) HeLa-GFP-LC3 cells were transfected with either control or αSNAP-specific siRNAs and formation of autophagosomes was visualized by confocal microscopy analysis of GFP fluorescence in fixed cells 72 h post-transfection. Data in this and other figures are presented as mean ± SEM of three independent experiments. *p
    Anti αsnap, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti αsnap/product/Abcam
    Average 86 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    anti αsnap - by Bioz Stars, 2020-08
    86/100 stars
      Buy from Supplier

    Image Search Results


    Figure 1. siRNA-mediated downregulation of αSNAP alters the autophagic flux. ( A ) SK-CO15 cells were transfected with either control or two different αSNAP-specific siRNA duplexes (D1 and D2). Expression of αSNAP and autophagic markers, LC3, NBR1 and p62 in total-cell lysates was determined by immunoblotting. ( B ) siRNA depletion of αSNAP was performed in either control SK-CO15 cells (SK-neo) or cells with stable expression of siRNA-resistant bovine αSNAP (SK-αSNAP). Expression of αSNAP and autophagic markers in cell lysates was determined by immunoblotting 48 h post-transfection. ( C and D ) HeLa-GFP-LC3 cells were transfected with either control or αSNAP-specific siRNAs and formation of autophagosomes was visualized by confocal microscopy analysis of GFP fluorescence in fixed cells 72 h post-transfection. Data in this and other figures are presented as mean ± SEM of three independent experiments. *p

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 1. siRNA-mediated downregulation of αSNAP alters the autophagic flux. ( A ) SK-CO15 cells were transfected with either control or two different αSNAP-specific siRNA duplexes (D1 and D2). Expression of αSNAP and autophagic markers, LC3, NBR1 and p62 in total-cell lysates was determined by immunoblotting. ( B ) siRNA depletion of αSNAP was performed in either control SK-CO15 cells (SK-neo) or cells with stable expression of siRNA-resistant bovine αSNAP (SK-αSNAP). Expression of αSNAP and autophagic markers in cell lysates was determined by immunoblotting 48 h post-transfection. ( C and D ) HeLa-GFP-LC3 cells were transfected with either control or αSNAP-specific siRNAs and formation of autophagosomes was visualized by confocal microscopy analysis of GFP fluorescence in fixed cells 72 h post-transfection. Data in this and other figures are presented as mean ± SEM of three independent experiments. *p

    Article Snippet: Since our results suggest a functional link between Golgi fragmentation and induction of autophagy in αSNAP-depleted epithelia, it is important to understand how loss of this membrane fusion protein can disrupt the Golgi architecture. αSNAP is known to be associated with two distinct SNARE complexes involving either syntaxin-5 , or syntaxin-18 , that, respectively, mediate the anterograde or retrograde vesicle trafficking between the ER and the Golgi.

    Techniques: Transfection, Expressing, Confocal Microscopy, Fluorescence

    Figure 10. Enhanced autophagy in αSNAP-depleted epithelial cells does not depend on BNIP1. ( A and B ) SK-CO15 cells were subjected to sequential transfections with one of the following siRNA pairs: control-control, control-BNIP1, control-αSNAP and BNIP1-αSNAP. Expression of targeted proteins and autophagic markers was determined by immunoblotting at 48 h after the second transfection. #p

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 10. Enhanced autophagy in αSNAP-depleted epithelial cells does not depend on BNIP1. ( A and B ) SK-CO15 cells were subjected to sequential transfections with one of the following siRNA pairs: control-control, control-BNIP1, control-αSNAP and BNIP1-αSNAP. Expression of targeted proteins and autophagic markers was determined by immunoblotting at 48 h after the second transfection. #p

    Article Snippet: Since our results suggest a functional link between Golgi fragmentation and induction of autophagy in αSNAP-depleted epithelia, it is important to understand how loss of this membrane fusion protein can disrupt the Golgi architecture. αSNAP is known to be associated with two distinct SNARE complexes involving either syntaxin-5 , or syntaxin-18 , that, respectively, mediate the anterograde or retrograde vesicle trafficking between the ER and the Golgi.

    Techniques: Transfection, Expressing

    Figure 5. Loss of αSNAP triggers fragmentation of the Golgi in parallel to autophagy induction. Control and αSNAP-depleted HeLa-GFP-LC3 cells were immunofluorescence labeled for Golgi markers Giantin and TGN46 (red) at 72 h post-transfection. Control cells are characterized by the compact perinuclear Golgi complex, whereas αSNAP depletion results in a dramatic fragmentation of the Golgi (arrows) and appearance of Golgi markers in LC3-positive autophagosomes (inserts). Scale bar, 10 µm.

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 5. Loss of αSNAP triggers fragmentation of the Golgi in parallel to autophagy induction. Control and αSNAP-depleted HeLa-GFP-LC3 cells were immunofluorescence labeled for Golgi markers Giantin and TGN46 (red) at 72 h post-transfection. Control cells are characterized by the compact perinuclear Golgi complex, whereas αSNAP depletion results in a dramatic fragmentation of the Golgi (arrows) and appearance of Golgi markers in LC3-positive autophagosomes (inserts). Scale bar, 10 µm.

    Article Snippet: Since our results suggest a functional link between Golgi fragmentation and induction of autophagy in αSNAP-depleted epithelia, it is important to understand how loss of this membrane fusion protein can disrupt the Golgi architecture. αSNAP is known to be associated with two distinct SNARE complexes involving either syntaxin-5 , or syntaxin-18 , that, respectively, mediate the anterograde or retrograde vesicle trafficking between the ER and the Golgi.

    Techniques: Immunofluorescence, Labeling, Transfection

    Figure 7. Loss of αSNAP decreases expression of Brefeldin-sensitive guanine nucleotide exchange factors. ( A ) Expression of Brefeldin-sensitive exchange factors GBF1, BIG1 and BIG2 was examined in control and αSNAP-depleted SK-CO15 cells 48 h after siRNA transfection. ( B ) Effect of αSNAP knockdown on localization of GBF1 (red) in SK-CO15 cells was analyzed by immunofluorescence labeling and confocal microscopy. Scale bar, 20 µm.

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 7. Loss of αSNAP decreases expression of Brefeldin-sensitive guanine nucleotide exchange factors. ( A ) Expression of Brefeldin-sensitive exchange factors GBF1, BIG1 and BIG2 was examined in control and αSNAP-depleted SK-CO15 cells 48 h after siRNA transfection. ( B ) Effect of αSNAP knockdown on localization of GBF1 (red) in SK-CO15 cells was analyzed by immunofluorescence labeling and confocal microscopy. Scale bar, 20 µm.

    Article Snippet: Since our results suggest a functional link between Golgi fragmentation and induction of autophagy in αSNAP-depleted epithelia, it is important to understand how loss of this membrane fusion protein can disrupt the Golgi architecture. αSNAP is known to be associated with two distinct SNARE complexes involving either syntaxin-5 , or syntaxin-18 , that, respectively, mediate the anterograde or retrograde vesicle trafficking between the ER and the Golgi.

    Techniques: Expressing, Transfection, Immunofluorescence, Labeling, Confocal Microscopy

    Figure 4. Atg5 and Atg7 play roles in induction of autophagy caused by downregulation of αSNAP. ( A and B ) SK-CO15 cells were subjected to sequential transfections with one of the following siRNA pairs: control-control, control-Atg5, control-Atg7, control-αSNAP, Atg5-αSNAP or Atg7-αSNAP. Expression of LC3, αSNAP, Atg5 and Atg7 was determined by immunoblotting 48 h after the second transfection. #p

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 4. Atg5 and Atg7 play roles in induction of autophagy caused by downregulation of αSNAP. ( A and B ) SK-CO15 cells were subjected to sequential transfections with one of the following siRNA pairs: control-control, control-Atg5, control-Atg7, control-αSNAP, Atg5-αSNAP or Atg7-αSNAP. Expression of LC3, αSNAP, Atg5 and Atg7 was determined by immunoblotting 48 h after the second transfection. #p

    Article Snippet: Since our results suggest a functional link between Golgi fragmentation and induction of autophagy in αSNAP-depleted epithelia, it is important to understand how loss of this membrane fusion protein can disrupt the Golgi architecture. αSNAP is known to be associated with two distinct SNARE complexes involving either syntaxin-5 , or syntaxin-18 , that, respectively, mediate the anterograde or retrograde vesicle trafficking between the ER and the Golgi.

    Techniques: Transfection, Expressing

    Figure 2. Lysosomal inhibitors exaggerate LC3 conjugation and accumulation of autophagosomes in αSNAP-depleted epithelial cells. SK-CO15 ( A and B ) or HeLa-GFP-LC3 ( C ) cells transfected with either control or αSNAP duplex 1 siRNAs were treated for 4 h with either vehicle or lysosomal inhibitors bafilomycin A (0.2 µM) or chloroquine (100 µM). Expression of LC3-II was determined in SK-CO15 cells by immunoblotting, whereas formation of autophagosomes was examined in HeLa-GFP-LC3 cells by fluorescence microscopy. **p

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 2. Lysosomal inhibitors exaggerate LC3 conjugation and accumulation of autophagosomes in αSNAP-depleted epithelial cells. SK-CO15 ( A and B ) or HeLa-GFP-LC3 ( C ) cells transfected with either control or αSNAP duplex 1 siRNAs were treated for 4 h with either vehicle or lysosomal inhibitors bafilomycin A (0.2 µM) or chloroquine (100 µM). Expression of LC3-II was determined in SK-CO15 cells by immunoblotting, whereas formation of autophagosomes was examined in HeLa-GFP-LC3 cells by fluorescence microscopy. **p

    Article Snippet: Since our results suggest a functional link between Golgi fragmentation and induction of autophagy in αSNAP-depleted epithelia, it is important to understand how loss of this membrane fusion protein can disrupt the Golgi architecture. αSNAP is known to be associated with two distinct SNARE complexes involving either syntaxin-5 , or syntaxin-18 , that, respectively, mediate the anterograde or retrograde vesicle trafficking between the ER and the Golgi.

    Techniques: Conjugation Assay, Transfection, Expressing, Fluorescence, Microscopy

    Figure 9. Bif-1 is involved in the enhanced autophagy caused by downregulation of αSNAP. SK-CO15 ( A and B ) and HeLa-GFP-LC3 cells were subjected to sequential transfections with one of the following siRNA pairs: control-control, control-Bif-1, control-αSNAP and Bif-1-αSNAP. Levels of LC3 and Bif-1, as well as accumulation of autophagosomes, were determined by immunoblotting and fluorescence microscopy, respectively. #p

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 9. Bif-1 is involved in the enhanced autophagy caused by downregulation of αSNAP. SK-CO15 ( A and B ) and HeLa-GFP-LC3 cells were subjected to sequential transfections with one of the following siRNA pairs: control-control, control-Bif-1, control-αSNAP and Bif-1-αSNAP. Levels of LC3 and Bif-1, as well as accumulation of autophagosomes, were determined by immunoblotting and fluorescence microscopy, respectively. #p

    Article Snippet: Since our results suggest a functional link between Golgi fragmentation and induction of autophagy in αSNAP-depleted epithelia, it is important to understand how loss of this membrane fusion protein can disrupt the Golgi architecture. αSNAP is known to be associated with two distinct SNARE complexes involving either syntaxin-5 , or syntaxin-18 , that, respectively, mediate the anterograde or retrograde vesicle trafficking between the ER and the Golgi.

    Techniques: Transfection, Fluorescence, Microscopy

    Figure 3. Loss of αSNAP decreases expression of mTOR and its downstream effector, 4E-BP1. SK-CO15 cells were transfected with either control or αSNAP-specific siRNAs. Expression and phosphorylation of mTOR and its downstream effectors p70 S6 kinase and 4E-BP1 in total-cell lysates was determined at different times post-transfection.

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 3. Loss of αSNAP decreases expression of mTOR and its downstream effector, 4E-BP1. SK-CO15 cells were transfected with either control or αSNAP-specific siRNAs. Expression and phosphorylation of mTOR and its downstream effectors p70 S6 kinase and 4E-BP1 in total-cell lysates was determined at different times post-transfection.

    Article Snippet: Since our results suggest a functional link between Golgi fragmentation and induction of autophagy in αSNAP-depleted epithelia, it is important to understand how loss of this membrane fusion protein can disrupt the Golgi architecture. αSNAP is known to be associated with two distinct SNARE complexes involving either syntaxin-5 , or syntaxin-18 , that, respectively, mediate the anterograde or retrograde vesicle trafficking between the ER and the Golgi.

    Techniques: Expressing, Transfection

    Figure 1. siRNA-mediated downregulation of αSNAP alters the autophagic flux. ( A ) SK-CO15 cells were transfected with either control or two different αSNAP-specific siRNA duplexes (D1 and D2). Expression of αSNAP and autophagic markers, LC3, NBR1 and p62 in total-cell lysates was determined by immunoblotting. ( B ) siRNA depletion of αSNAP was performed in either control SK-CO15 cells (SK-neo) or cells with stable expression of siRNA-resistant bovine αSNAP (SK-αSNAP). Expression of αSNAP and autophagic markers in cell lysates was determined by immunoblotting 48 h post-transfection. ( C and D ) HeLa-GFP-LC3 cells were transfected with either control or αSNAP-specific siRNAs and formation of autophagosomes was visualized by confocal microscopy analysis of GFP fluorescence in fixed cells 72 h post-transfection. Data in this and other figures are presented as mean ± SEM of three independent experiments. *p

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 1. siRNA-mediated downregulation of αSNAP alters the autophagic flux. ( A ) SK-CO15 cells were transfected with either control or two different αSNAP-specific siRNA duplexes (D1 and D2). Expression of αSNAP and autophagic markers, LC3, NBR1 and p62 in total-cell lysates was determined by immunoblotting. ( B ) siRNA depletion of αSNAP was performed in either control SK-CO15 cells (SK-neo) or cells with stable expression of siRNA-resistant bovine αSNAP (SK-αSNAP). Expression of αSNAP and autophagic markers in cell lysates was determined by immunoblotting 48 h post-transfection. ( C and D ) HeLa-GFP-LC3 cells were transfected with either control or αSNAP-specific siRNAs and formation of autophagosomes was visualized by confocal microscopy analysis of GFP fluorescence in fixed cells 72 h post-transfection. Data in this and other figures are presented as mean ± SEM of three independent experiments. *p

    Article Snippet: The following primary monoclonal (mAb) and polyclonal antibodies (pAb) were used to detect the signaling and autophagy-related proteins: anti-αSNAP, p150, NBR1 and GFP mAbs (Abcam); anti-cleaved PARP, active caspase-7, Atg5, Atg7, LC3, beclin-1, phospho-mTOR, p70 S6 kinase, phospho-p70 S6 kinase.

    Techniques: Transfection, Expressing, Confocal Microscopy, Fluorescence

    Figure 10. Enhanced autophagy in αSNAP-depleted epithelial cells does not depend on BNIP1. ( A and B ) SK-CO15 cells were subjected to sequential transfections with one of the following siRNA pairs: control-control, control-BNIP1, control-αSNAP and BNIP1-αSNAP. Expression of targeted proteins and autophagic markers was determined by immunoblotting at 48 h after the second transfection. #p

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 10. Enhanced autophagy in αSNAP-depleted epithelial cells does not depend on BNIP1. ( A and B ) SK-CO15 cells were subjected to sequential transfections with one of the following siRNA pairs: control-control, control-BNIP1, control-αSNAP and BNIP1-αSNAP. Expression of targeted proteins and autophagic markers was determined by immunoblotting at 48 h after the second transfection. #p

    Article Snippet: The following primary monoclonal (mAb) and polyclonal antibodies (pAb) were used to detect the signaling and autophagy-related proteins: anti-αSNAP, p150, NBR1 and GFP mAbs (Abcam); anti-cleaved PARP, active caspase-7, Atg5, Atg7, LC3, beclin-1, phospho-mTOR, p70 S6 kinase, phospho-p70 S6 kinase.

    Techniques: Transfection, Expressing

    Figure 5. Loss of αSNAP triggers fragmentation of the Golgi in parallel to autophagy induction. Control and αSNAP-depleted HeLa-GFP-LC3 cells were immunofluorescence labeled for Golgi markers Giantin and TGN46 (red) at 72 h post-transfection. Control cells are characterized by the compact perinuclear Golgi complex, whereas αSNAP depletion results in a dramatic fragmentation of the Golgi (arrows) and appearance of Golgi markers in LC3-positive autophagosomes (inserts). Scale bar, 10 µm.

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 5. Loss of αSNAP triggers fragmentation of the Golgi in parallel to autophagy induction. Control and αSNAP-depleted HeLa-GFP-LC3 cells were immunofluorescence labeled for Golgi markers Giantin and TGN46 (red) at 72 h post-transfection. Control cells are characterized by the compact perinuclear Golgi complex, whereas αSNAP depletion results in a dramatic fragmentation of the Golgi (arrows) and appearance of Golgi markers in LC3-positive autophagosomes (inserts). Scale bar, 10 µm.

    Article Snippet: The following primary monoclonal (mAb) and polyclonal antibodies (pAb) were used to detect the signaling and autophagy-related proteins: anti-αSNAP, p150, NBR1 and GFP mAbs (Abcam); anti-cleaved PARP, active caspase-7, Atg5, Atg7, LC3, beclin-1, phospho-mTOR, p70 S6 kinase, phospho-p70 S6 kinase.

    Techniques: Immunofluorescence, Labeling, Transfection

    Figure 7. Loss of αSNAP decreases expression of Brefeldin-sensitive guanine nucleotide exchange factors. ( A ) Expression of Brefeldin-sensitive exchange factors GBF1, BIG1 and BIG2 was examined in control and αSNAP-depleted SK-CO15 cells 48 h after siRNA transfection. ( B ) Effect of αSNAP knockdown on localization of GBF1 (red) in SK-CO15 cells was analyzed by immunofluorescence labeling and confocal microscopy. Scale bar, 20 µm.

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 7. Loss of αSNAP decreases expression of Brefeldin-sensitive guanine nucleotide exchange factors. ( A ) Expression of Brefeldin-sensitive exchange factors GBF1, BIG1 and BIG2 was examined in control and αSNAP-depleted SK-CO15 cells 48 h after siRNA transfection. ( B ) Effect of αSNAP knockdown on localization of GBF1 (red) in SK-CO15 cells was analyzed by immunofluorescence labeling and confocal microscopy. Scale bar, 20 µm.

    Article Snippet: The following primary monoclonal (mAb) and polyclonal antibodies (pAb) were used to detect the signaling and autophagy-related proteins: anti-αSNAP, p150, NBR1 and GFP mAbs (Abcam); anti-cleaved PARP, active caspase-7, Atg5, Atg7, LC3, beclin-1, phospho-mTOR, p70 S6 kinase, phospho-p70 S6 kinase.

    Techniques: Expressing, Transfection, Immunofluorescence, Labeling, Confocal Microscopy

    Figure 4. Atg5 and Atg7 play roles in induction of autophagy caused by downregulation of αSNAP. ( A and B ) SK-CO15 cells were subjected to sequential transfections with one of the following siRNA pairs: control-control, control-Atg5, control-Atg7, control-αSNAP, Atg5-αSNAP or Atg7-αSNAP. Expression of LC3, αSNAP, Atg5 and Atg7 was determined by immunoblotting 48 h after the second transfection. #p

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 4. Atg5 and Atg7 play roles in induction of autophagy caused by downregulation of αSNAP. ( A and B ) SK-CO15 cells were subjected to sequential transfections with one of the following siRNA pairs: control-control, control-Atg5, control-Atg7, control-αSNAP, Atg5-αSNAP or Atg7-αSNAP. Expression of LC3, αSNAP, Atg5 and Atg7 was determined by immunoblotting 48 h after the second transfection. #p

    Article Snippet: The following primary monoclonal (mAb) and polyclonal antibodies (pAb) were used to detect the signaling and autophagy-related proteins: anti-αSNAP, p150, NBR1 and GFP mAbs (Abcam); anti-cleaved PARP, active caspase-7, Atg5, Atg7, LC3, beclin-1, phospho-mTOR, p70 S6 kinase, phospho-p70 S6 kinase.

    Techniques: Transfection, Expressing

    Figure 2. Lysosomal inhibitors exaggerate LC3 conjugation and accumulation of autophagosomes in αSNAP-depleted epithelial cells. SK-CO15 ( A and B ) or HeLa-GFP-LC3 ( C ) cells transfected with either control or αSNAP duplex 1 siRNAs were treated for 4 h with either vehicle or lysosomal inhibitors bafilomycin A (0.2 µM) or chloroquine (100 µM). Expression of LC3-II was determined in SK-CO15 cells by immunoblotting, whereas formation of autophagosomes was examined in HeLa-GFP-LC3 cells by fluorescence microscopy. **p

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 2. Lysosomal inhibitors exaggerate LC3 conjugation and accumulation of autophagosomes in αSNAP-depleted epithelial cells. SK-CO15 ( A and B ) or HeLa-GFP-LC3 ( C ) cells transfected with either control or αSNAP duplex 1 siRNAs were treated for 4 h with either vehicle or lysosomal inhibitors bafilomycin A (0.2 µM) or chloroquine (100 µM). Expression of LC3-II was determined in SK-CO15 cells by immunoblotting, whereas formation of autophagosomes was examined in HeLa-GFP-LC3 cells by fluorescence microscopy. **p

    Article Snippet: The following primary monoclonal (mAb) and polyclonal antibodies (pAb) were used to detect the signaling and autophagy-related proteins: anti-αSNAP, p150, NBR1 and GFP mAbs (Abcam); anti-cleaved PARP, active caspase-7, Atg5, Atg7, LC3, beclin-1, phospho-mTOR, p70 S6 kinase, phospho-p70 S6 kinase.

    Techniques: Conjugation Assay, Transfection, Expressing, Fluorescence, Microscopy

    Figure 9. Bif-1 is involved in the enhanced autophagy caused by downregulation of αSNAP. SK-CO15 ( A and B ) and HeLa-GFP-LC3 cells were subjected to sequential transfections with one of the following siRNA pairs: control-control, control-Bif-1, control-αSNAP and Bif-1-αSNAP. Levels of LC3 and Bif-1, as well as accumulation of autophagosomes, were determined by immunoblotting and fluorescence microscopy, respectively. #p

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 9. Bif-1 is involved in the enhanced autophagy caused by downregulation of αSNAP. SK-CO15 ( A and B ) and HeLa-GFP-LC3 cells were subjected to sequential transfections with one of the following siRNA pairs: control-control, control-Bif-1, control-αSNAP and Bif-1-αSNAP. Levels of LC3 and Bif-1, as well as accumulation of autophagosomes, were determined by immunoblotting and fluorescence microscopy, respectively. #p

    Article Snippet: The following primary monoclonal (mAb) and polyclonal antibodies (pAb) were used to detect the signaling and autophagy-related proteins: anti-αSNAP, p150, NBR1 and GFP mAbs (Abcam); anti-cleaved PARP, active caspase-7, Atg5, Atg7, LC3, beclin-1, phospho-mTOR, p70 S6 kinase, phospho-p70 S6 kinase.

    Techniques: Transfection, Fluorescence, Microscopy

    Figure 3. Loss of αSNAP decreases expression of mTOR and its downstream effector, 4E-BP1. SK-CO15 cells were transfected with either control or αSNAP-specific siRNAs. Expression and phosphorylation of mTOR and its downstream effectors p70 S6 kinase and 4E-BP1 in total-cell lysates was determined at different times post-transfection.

    Journal: Cell Cycle

    Article Title: Loss of a membrane trafficking protein ?SNAP induces non-canonical autophagy in human epithelia

    doi: 10.4161/cc.22885

    Figure Lengend Snippet: Figure 3. Loss of αSNAP decreases expression of mTOR and its downstream effector, 4E-BP1. SK-CO15 cells were transfected with either control or αSNAP-specific siRNAs. Expression and phosphorylation of mTOR and its downstream effectors p70 S6 kinase and 4E-BP1 in total-cell lysates was determined at different times post-transfection.

    Article Snippet: The following primary monoclonal (mAb) and polyclonal antibodies (pAb) were used to detect the signaling and autophagy-related proteins: anti-αSNAP, p150, NBR1 and GFP mAbs (Abcam); anti-cleaved PARP, active caspase-7, Atg5, Atg7, LC3, beclin-1, phospho-mTOR, p70 S6 kinase, phospho-p70 S6 kinase.

    Techniques: Expressing, Transfection

    Downregulation of GBF1 expression phenocopies the effects of αSNAP depletion on epithelial junctions. Immunofluorescence labeling shows formation of normal β-catenin-based AJs, occludin/claudin-4-based TJs and well-organized perinuclear Golgi ribbon (arrows) in control SK-CO15 cell monolayers. In contrast, GBF1-depleted cells display a defective AJ/TJ assembly, intracellular localization of junctional proteins and dispersed Golgi (arrowheads) on day 4 post-transfection. Scale bar, 20 µm.

    Journal: PLoS ONE

    Article Title: A Membrane Fusion Protein ?SNAP Is a Novel Regulator of Epithelial Apical Junctions

    doi: 10.1371/journal.pone.0034320

    Figure Lengend Snippet: Downregulation of GBF1 expression phenocopies the effects of αSNAP depletion on epithelial junctions. Immunofluorescence labeling shows formation of normal β-catenin-based AJs, occludin/claudin-4-based TJs and well-organized perinuclear Golgi ribbon (arrows) in control SK-CO15 cell monolayers. In contrast, GBF1-depleted cells display a defective AJ/TJ assembly, intracellular localization of junctional proteins and dispersed Golgi (arrowheads) on day 4 post-transfection. Scale bar, 20 µm.

    Article Snippet: Antibodies and other reagents The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect junctional, signaling and vesicle trafficking proteins: anti-αSNAP mAb (Abcam) anti-occludin, ZO-1, claudin-1, claudin-4, and JAM-A mAbs and pAbs (Invitrogen, Carlsbad, CA); anti-cleaved PARP, active caspase-3, active caspase-7 and GAPDH pAbs (Cell Signaling); anti-E-cadherin, β-catenin, GM-130 and NSF mAbs (BD Biosciences, San Jose, CA); anti-ERGIC53 mAb (Enzo Life Sciences, Plymouth Meetings, PA); anti-BIG1 and Giantin pAbs (Covance, Princeton, NJ); anti-β-catenin pAb (Sigma-Aldrich, St. Louis, MO); anti-α-catenin mAb (Epitomics, Burlingame, CA).

    Techniques: Expressing, Immunofluorescence, Labeling, Transfection

    αSNAP is enriched at epithelial junctions in vitro and in vivo. ( A ) Dual immunofluorescence labeling and confocal microscopy show colocalization (arrows) of αSNAP (red) and tight junction protein occludin (green) in confluent T84 cell monolayers and normal human colonic mucosa. ( B ) Immunofluorescence images show predominantly-perinuclear localization of αSNAP in calcium-depleted SK-CO15 cells (arrowheads) and its rapid translocation to newly-formed AJs in calcium-repleted cells (arrows). Scale bar, 10 µM.

    Journal: PLoS ONE

    Article Title: A Membrane Fusion Protein ?SNAP Is a Novel Regulator of Epithelial Apical Junctions

    doi: 10.1371/journal.pone.0034320

    Figure Lengend Snippet: αSNAP is enriched at epithelial junctions in vitro and in vivo. ( A ) Dual immunofluorescence labeling and confocal microscopy show colocalization (arrows) of αSNAP (red) and tight junction protein occludin (green) in confluent T84 cell monolayers and normal human colonic mucosa. ( B ) Immunofluorescence images show predominantly-perinuclear localization of αSNAP in calcium-depleted SK-CO15 cells (arrowheads) and its rapid translocation to newly-formed AJs in calcium-repleted cells (arrows). Scale bar, 10 µM.

    Article Snippet: Antibodies and other reagents The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect junctional, signaling and vesicle trafficking proteins: anti-αSNAP mAb (Abcam) anti-occludin, ZO-1, claudin-1, claudin-4, and JAM-A mAbs and pAbs (Invitrogen, Carlsbad, CA); anti-cleaved PARP, active caspase-3, active caspase-7 and GAPDH pAbs (Cell Signaling); anti-E-cadherin, β-catenin, GM-130 and NSF mAbs (BD Biosciences, San Jose, CA); anti-ERGIC53 mAb (Enzo Life Sciences, Plymouth Meetings, PA); anti-BIG1 and Giantin pAbs (Covance, Princeton, NJ); anti-β-catenin pAb (Sigma-Aldrich, St. Louis, MO); anti-α-catenin mAb (Epitomics, Burlingame, CA).

    Techniques: In Vitro, In Vivo, Immunofluorescence, Labeling, Confocal Microscopy, Translocation Assay

    Downregulation of NSF does not phenocopy the major effects of αSNAP knockdown on epithelial junctions. ( A ) Immunoblotting analysis shows that NSF-specific siRNA efficiently decreases expression of the targeted protein without affecting αSNAP and p120 catenin protein levels. ( B ) TEER measurements demonstrate that NSF-depletion significantly attenuates formation of the paracellular barrier in SK-CO15 cell monolayers; *p

    Journal: PLoS ONE

    Article Title: A Membrane Fusion Protein ?SNAP Is a Novel Regulator of Epithelial Apical Junctions

    doi: 10.1371/journal.pone.0034320

    Figure Lengend Snippet: Downregulation of NSF does not phenocopy the major effects of αSNAP knockdown on epithelial junctions. ( A ) Immunoblotting analysis shows that NSF-specific siRNA efficiently decreases expression of the targeted protein without affecting αSNAP and p120 catenin protein levels. ( B ) TEER measurements demonstrate that NSF-depletion significantly attenuates formation of the paracellular barrier in SK-CO15 cell monolayers; *p

    Article Snippet: Antibodies and other reagents The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect junctional, signaling and vesicle trafficking proteins: anti-αSNAP mAb (Abcam) anti-occludin, ZO-1, claudin-1, claudin-4, and JAM-A mAbs and pAbs (Invitrogen, Carlsbad, CA); anti-cleaved PARP, active caspase-3, active caspase-7 and GAPDH pAbs (Cell Signaling); anti-E-cadherin, β-catenin, GM-130 and NSF mAbs (BD Biosciences, San Jose, CA); anti-ERGIC53 mAb (Enzo Life Sciences, Plymouth Meetings, PA); anti-BIG1 and Giantin pAbs (Covance, Princeton, NJ); anti-β-catenin pAb (Sigma-Aldrich, St. Louis, MO); anti-α-catenin mAb (Epitomics, Burlingame, CA).

    Techniques: Expressing

    αSNAP depletion induces fragmentation of the Golgi and ERGIC. Control and αSNAP-depleted SK-CO15 cells were immunofluorescence labeled for β-catenin (green) with either Golgi marker GM130, or ERGIC marker ERGIC53 (red) on day 3 post-transfection. Control cells show perinuclear tubular Golgi complexes and well-organized compact ERGIC (arrows). By contrast, αSNAP depletion results in a dramatic dispersion of both perinuclear Golgi and ERGIC structures (arrowheads). Scale bar, 10 µm.

    Journal: PLoS ONE

    Article Title: A Membrane Fusion Protein ?SNAP Is a Novel Regulator of Epithelial Apical Junctions

    doi: 10.1371/journal.pone.0034320

    Figure Lengend Snippet: αSNAP depletion induces fragmentation of the Golgi and ERGIC. Control and αSNAP-depleted SK-CO15 cells were immunofluorescence labeled for β-catenin (green) with either Golgi marker GM130, or ERGIC marker ERGIC53 (red) on day 3 post-transfection. Control cells show perinuclear tubular Golgi complexes and well-organized compact ERGIC (arrows). By contrast, αSNAP depletion results in a dramatic dispersion of both perinuclear Golgi and ERGIC structures (arrowheads). Scale bar, 10 µm.

    Article Snippet: Antibodies and other reagents The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect junctional, signaling and vesicle trafficking proteins: anti-αSNAP mAb (Abcam) anti-occludin, ZO-1, claudin-1, claudin-4, and JAM-A mAbs and pAbs (Invitrogen, Carlsbad, CA); anti-cleaved PARP, active caspase-3, active caspase-7 and GAPDH pAbs (Cell Signaling); anti-E-cadherin, β-catenin, GM-130 and NSF mAbs (BD Biosciences, San Jose, CA); anti-ERGIC53 mAb (Enzo Life Sciences, Plymouth Meetings, PA); anti-BIG1 and Giantin pAbs (Covance, Princeton, NJ); anti-β-catenin pAb (Sigma-Aldrich, St. Louis, MO); anti-α-catenin mAb (Epitomics, Burlingame, CA).

    Techniques: Immunofluorescence, Labeling, Marker, Transfection

    siRNA-mediated depletion of αSNAP prevents formation of the paracellular barrier and AJ/TJ assembly, and selectively downregulates p120 catenin and E-cadherin expression. ( A ) Immunoblotting analysis shows that two different αSNAP-specific siRNA duplexes (D1 D2) dramatically decrease its protein expression in SK-CO15 cells on day 3 post-transfection. ( B ) TEER measurements demonstrate that αSNAP-depleted SK-CO15 cell monolayers fail to develop the paracellular barrier. ( C ) Immunofluorescence labeling shows formation of normal β-catenin-based AJs and occludin-based TJs (arrows) in control SK-CO15 cell monolayers on day 4 post-transfection. By contrast, αSNAP-depleted cells have significant intracellular accumulation of β-catenin and fragmented TJ strands (arrowheads). ( D ) Immunoblotting analysis shows that selective αSNAP depletion increases the level of TJ proteins but dramatically downregulates p120 catenin and E-cadherin expression in SK-CO15 cells on day 4 post-transfection; *p

    Journal: PLoS ONE

    Article Title: A Membrane Fusion Protein ?SNAP Is a Novel Regulator of Epithelial Apical Junctions

    doi: 10.1371/journal.pone.0034320

    Figure Lengend Snippet: siRNA-mediated depletion of αSNAP prevents formation of the paracellular barrier and AJ/TJ assembly, and selectively downregulates p120 catenin and E-cadherin expression. ( A ) Immunoblotting analysis shows that two different αSNAP-specific siRNA duplexes (D1 D2) dramatically decrease its protein expression in SK-CO15 cells on day 3 post-transfection. ( B ) TEER measurements demonstrate that αSNAP-depleted SK-CO15 cell monolayers fail to develop the paracellular barrier. ( C ) Immunofluorescence labeling shows formation of normal β-catenin-based AJs and occludin-based TJs (arrows) in control SK-CO15 cell monolayers on day 4 post-transfection. By contrast, αSNAP-depleted cells have significant intracellular accumulation of β-catenin and fragmented TJ strands (arrowheads). ( D ) Immunoblotting analysis shows that selective αSNAP depletion increases the level of TJ proteins but dramatically downregulates p120 catenin and E-cadherin expression in SK-CO15 cells on day 4 post-transfection; *p

    Article Snippet: Antibodies and other reagents The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect junctional, signaling and vesicle trafficking proteins: anti-αSNAP mAb (Abcam) anti-occludin, ZO-1, claudin-1, claudin-4, and JAM-A mAbs and pAbs (Invitrogen, Carlsbad, CA); anti-cleaved PARP, active caspase-3, active caspase-7 and GAPDH pAbs (Cell Signaling); anti-E-cadherin, β-catenin, GM-130 and NSF mAbs (BD Biosciences, San Jose, CA); anti-ERGIC53 mAb (Enzo Life Sciences, Plymouth Meetings, PA); anti-BIG1 and Giantin pAbs (Covance, Princeton, NJ); anti-β-catenin pAb (Sigma-Aldrich, St. Louis, MO); anti-α-catenin mAb (Epitomics, Burlingame, CA).

    Techniques: Expressing, Transfection, Immunofluorescence, Labeling

    GBF1, but not other Golgi GEFs, regulates barrier functions of the intestinal epithelial junctions. ( A ) Immunoblotting analysis demonstrates that two different αSNAP-specific siRNA duplexes significantly decrease expression of Golgi GEFs, GBF1, BIG1 and BIG2 in SK-CO15 cells on days 2 and 3 post-transfection. ( B,C ) SK-CO15 cells were transfected with either control GBF1, BIG1, or BIG2, siRNAs and examined on day 4 post-transfection. Immunoblotting analysis demonstrates selective down regulation of individual GEFs by their gene-specific siRNAs. Depletion of GBF1, but not BIG1 or BIG2, decreased p120 catenin expression ( B ), dramatically inhibited development of the paracellular barrier ( C ) and induced apoptosis ( B ); *p

    Journal: PLoS ONE

    Article Title: A Membrane Fusion Protein ?SNAP Is a Novel Regulator of Epithelial Apical Junctions

    doi: 10.1371/journal.pone.0034320

    Figure Lengend Snippet: GBF1, but not other Golgi GEFs, regulates barrier functions of the intestinal epithelial junctions. ( A ) Immunoblotting analysis demonstrates that two different αSNAP-specific siRNA duplexes significantly decrease expression of Golgi GEFs, GBF1, BIG1 and BIG2 in SK-CO15 cells on days 2 and 3 post-transfection. ( B,C ) SK-CO15 cells were transfected with either control GBF1, BIG1, or BIG2, siRNAs and examined on day 4 post-transfection. Immunoblotting analysis demonstrates selective down regulation of individual GEFs by their gene-specific siRNAs. Depletion of GBF1, but not BIG1 or BIG2, decreased p120 catenin expression ( B ), dramatically inhibited development of the paracellular barrier ( C ) and induced apoptosis ( B ); *p

    Article Snippet: Antibodies and other reagents The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect junctional, signaling and vesicle trafficking proteins: anti-αSNAP mAb (Abcam) anti-occludin, ZO-1, claudin-1, claudin-4, and JAM-A mAbs and pAbs (Invitrogen, Carlsbad, CA); anti-cleaved PARP, active caspase-3, active caspase-7 and GAPDH pAbs (Cell Signaling); anti-E-cadherin, β-catenin, GM-130 and NSF mAbs (BD Biosciences, San Jose, CA); anti-ERGIC53 mAb (Enzo Life Sciences, Plymouth Meetings, PA); anti-BIG1 and Giantin pAbs (Covance, Princeton, NJ); anti-β-catenin pAb (Sigma-Aldrich, St. Louis, MO); anti-α-catenin mAb (Epitomics, Burlingame, CA).

    Techniques: Expressing, Transfection

    Junctional disassembly and apoptosis in αSNAP-depleted cells can be rescued by expression of siRNA-resistant bovine αSNAP. ( A ) Immunoblotting analysis shows preserved αSNAP level in SK-CO15 cells with stable expression of bovine αSNAP (SK-αSNAP) after siRNA depletion of endogenous human protein. Comparing to the control cells (SK-neo), such bovine αSNAP overexpression completely prevents induction of apoptosis ( A ), disruption of the paracellular barrier ( B ) and AJ/TJ disassembly ( C, arrowheads ) caused by loss of endogenous αSNAP; # p

    Journal: PLoS ONE

    Article Title: A Membrane Fusion Protein ?SNAP Is a Novel Regulator of Epithelial Apical Junctions

    doi: 10.1371/journal.pone.0034320

    Figure Lengend Snippet: Junctional disassembly and apoptosis in αSNAP-depleted cells can be rescued by expression of siRNA-resistant bovine αSNAP. ( A ) Immunoblotting analysis shows preserved αSNAP level in SK-CO15 cells with stable expression of bovine αSNAP (SK-αSNAP) after siRNA depletion of endogenous human protein. Comparing to the control cells (SK-neo), such bovine αSNAP overexpression completely prevents induction of apoptosis ( A ), disruption of the paracellular barrier ( B ) and AJ/TJ disassembly ( C, arrowheads ) caused by loss of endogenous αSNAP; # p

    Article Snippet: Antibodies and other reagents The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect junctional, signaling and vesicle trafficking proteins: anti-αSNAP mAb (Abcam) anti-occludin, ZO-1, claudin-1, claudin-4, and JAM-A mAbs and pAbs (Invitrogen, Carlsbad, CA); anti-cleaved PARP, active caspase-3, active caspase-7 and GAPDH pAbs (Cell Signaling); anti-E-cadherin, β-catenin, GM-130 and NSF mAbs (BD Biosciences, San Jose, CA); anti-ERGIC53 mAb (Enzo Life Sciences, Plymouth Meetings, PA); anti-BIG1 and Giantin pAbs (Covance, Princeton, NJ); anti-β-catenin pAb (Sigma-Aldrich, St. Louis, MO); anti-α-catenin mAb (Epitomics, Burlingame, CA).

    Techniques: Expressing, Over Expression

    Downregulation of p120 catenin expression phenocopies the effects of αSNAP depletion on epithelial junctions. ( A ) Immunoblotting analysis shows that p120 catenin-specific siRNA dramatically decreases p120-catenin and E-cadherin protein expression in SK-CO15 cells. ( B ) TEER measurements demonstrate that p120 catenin-depleted SK-CO15 cell monolayers do not develop the paracellular barrier on days 2–4 post-transfection. ( C ) Immunofluorescence labeling shows formation of normal β-catenin-based AJs and occludin/claudin-4-based TJs (arrows) in control SK-CO15 cell monolayers. In contrast, p120 catenin-depleted cells display a defective AJ/TJ assembly and intracellular localization of junctional proteins (arrowheads) on day 4 post-transfection; *p

    Journal: PLoS ONE

    Article Title: A Membrane Fusion Protein ?SNAP Is a Novel Regulator of Epithelial Apical Junctions

    doi: 10.1371/journal.pone.0034320

    Figure Lengend Snippet: Downregulation of p120 catenin expression phenocopies the effects of αSNAP depletion on epithelial junctions. ( A ) Immunoblotting analysis shows that p120 catenin-specific siRNA dramatically decreases p120-catenin and E-cadherin protein expression in SK-CO15 cells. ( B ) TEER measurements demonstrate that p120 catenin-depleted SK-CO15 cell monolayers do not develop the paracellular barrier on days 2–4 post-transfection. ( C ) Immunofluorescence labeling shows formation of normal β-catenin-based AJs and occludin/claudin-4-based TJs (arrows) in control SK-CO15 cell monolayers. In contrast, p120 catenin-depleted cells display a defective AJ/TJ assembly and intracellular localization of junctional proteins (arrowheads) on day 4 post-transfection; *p

    Article Snippet: Antibodies and other reagents The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect junctional, signaling and vesicle trafficking proteins: anti-αSNAP mAb (Abcam) anti-occludin, ZO-1, claudin-1, claudin-4, and JAM-A mAbs and pAbs (Invitrogen, Carlsbad, CA); anti-cleaved PARP, active caspase-3, active caspase-7 and GAPDH pAbs (Cell Signaling); anti-E-cadherin, β-catenin, GM-130 and NSF mAbs (BD Biosciences, San Jose, CA); anti-ERGIC53 mAb (Enzo Life Sciences, Plymouth Meetings, PA); anti-BIG1 and Giantin pAbs (Covance, Princeton, NJ); anti-β-catenin pAb (Sigma-Aldrich, St. Louis, MO); anti-α-catenin mAb (Epitomics, Burlingame, CA).

    Techniques: Expressing, Transfection, Immunofluorescence, Labeling

    Disruption of apical junctions and downregulation of p120 catenin expression in αSNAP-depleted cells are not mediated by apoptosis. ( A ) Immunoblotting analysis of cleaved PARP and active caspases 3 7 shows that αSNAP depletion causes significant cell apoptosis in SK-CO15 cells on day 4 post-transfection, which is prevented by inhibition of caspases with Z-VAD-fmk (50 µM). ( B–D ) To examine the role of apoptosis in junction disassembly, SK-CO15 cells were transfected with either control or αSNAP-specific siRNA (duplex 1), and one day later, were exposed to either vehicle or Z-VAD-fmk (50 µM) for 72 h. Inhibition of apoptosis does not prevent disruption of the paracellular barrier ( B ), disruption of AJs and TJs ( C , arrows) or down-regulation of p120 catenin expression ( D ) in αSNAP-depleted epithelial cells; *p

    Journal: PLoS ONE

    Article Title: A Membrane Fusion Protein ?SNAP Is a Novel Regulator of Epithelial Apical Junctions

    doi: 10.1371/journal.pone.0034320

    Figure Lengend Snippet: Disruption of apical junctions and downregulation of p120 catenin expression in αSNAP-depleted cells are not mediated by apoptosis. ( A ) Immunoblotting analysis of cleaved PARP and active caspases 3 7 shows that αSNAP depletion causes significant cell apoptosis in SK-CO15 cells on day 4 post-transfection, which is prevented by inhibition of caspases with Z-VAD-fmk (50 µM). ( B–D ) To examine the role of apoptosis in junction disassembly, SK-CO15 cells were transfected with either control or αSNAP-specific siRNA (duplex 1), and one day later, were exposed to either vehicle or Z-VAD-fmk (50 µM) for 72 h. Inhibition of apoptosis does not prevent disruption of the paracellular barrier ( B ), disruption of AJs and TJs ( C , arrows) or down-regulation of p120 catenin expression ( D ) in αSNAP-depleted epithelial cells; *p

    Article Snippet: Antibodies and other reagents The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect junctional, signaling and vesicle trafficking proteins: anti-αSNAP mAb (Abcam) anti-occludin, ZO-1, claudin-1, claudin-4, and JAM-A mAbs and pAbs (Invitrogen, Carlsbad, CA); anti-cleaved PARP, active caspase-3, active caspase-7 and GAPDH pAbs (Cell Signaling); anti-E-cadherin, β-catenin, GM-130 and NSF mAbs (BD Biosciences, San Jose, CA); anti-ERGIC53 mAb (Enzo Life Sciences, Plymouth Meetings, PA); anti-BIG1 and Giantin pAbs (Covance, Princeton, NJ); anti-β-catenin pAb (Sigma-Aldrich, St. Louis, MO); anti-α-catenin mAb (Epitomics, Burlingame, CA).

    Techniques: Expressing, Transfection, Inhibition