syntaxin 4 rabbit polyclonal  (Proteintech)

Journal: Traffic (Copenhagen, Denmark)

Article Title: Genetic evidence for an inhibitory role of tomosyn in insulin-stimulated GLUT4 exocytosis

doi: 10.1111/tra.12760

Figure Lengend Snippet: (A) Liposomes containing GLUT4 exocytic t-SNAREs (syntaxin-4 and SNAP-23) were incubated with or without 5 μM tomosyn-1 for one hour at 4 °C to form the t-SNARE/tomosyn-1 complex. Liposomes bearing the t-SNARE/tomosyn-1 complex were then incubated with or without 0.5 μM NSF, 1 μM α-SNAP, 2.5 mM ATP, and 5 mM MgCl2/EDTA at 37 °C for another hour. After flotation on a Nycodenz gradient, proteins bound to the liposomes were resolved on SDS-PAGE and stained with coomassie blue. Asterisk: α-SNAP co-migrated with syntaxin-4 on SDS-PAGE but its binding to t-SNARE liposomes was evident. (B) Coomassie blue-stained SDS-PAGE gel showing recombinant NSF and α-SNAP proteins used in this study.

Article Snippet: Primary antibodies used in immunoblotting included mouse monoclonal anti-tomosyn-1/2 antibodies (BD Biosciences, #611296), rabbit polyclonal anti-syntaxin-4 antibodies (Sigma, #S9924), mouse monoclonal anti-PPARγ antibodies (Santa Cruz Biotechnology, #sc-7273), anti-α-tubulin (DSHB, clone, #12G10), rabbit polyclonal anti-phospho-Akt (Ser473) antibodies (Cell Signaling Technology, #9271), rabbit polyclonal anti-Akt antibodies (Cell Signaling Technology, #9272), and mouse monoclonal anti-FLAG antibodies.

Techniques: Incubation, SDS Page, Staining, Binding Assay, Recombinant

Journal: Traffic (Copenhagen, Denmark)

Article Title: Genetic evidence for an inhibitory role of tomosyn in insulin-stimulated GLUT4 exocytosis

doi: 10.1111/tra.12760

Figure Lengend Snippet: (A) Diagram illustrating the reconstituted liposome fusion reactions. The t-SNARE liposomes containing syntaxin-4 and SNAP-23 were directed to fuse with VAMP2-bearing liposomes in the absence or presence of 5 μM tomosyn-1. Each fusion reaction contained 5 μM t-SNAREs, 1.5 μM v-SNARE, and 100 mg/mL Ficoll 70 as the crowding agent. To test the activities of NSF and α-SNAP, the following components were added to a fusion reaction: 0.5 μM NSF, 1 μM α-SNAP, 2.5 mM ATP, and 5 mM MgCl2 or EDTA. (B) Lipid mixing of the fusion reactions was measured using a FRET-based assay. Control: liposome fusion reactions without NSF or α-SNAP. (C) Lipid-mixing rates of the liposome fusion reactions shown in B. Data are presented as mean ± SD. n = 3. P values were calculated using Student’s t-test. ** P<0.01. n.s., P>0.05.

Article Snippet: Primary antibodies used in immunoblotting included mouse monoclonal anti-tomosyn-1/2 antibodies (BD Biosciences, #611296), rabbit polyclonal anti-syntaxin-4 antibodies (Sigma, #S9924), mouse monoclonal anti-PPARγ antibodies (Santa Cruz Biotechnology, #sc-7273), anti-α-tubulin (DSHB, clone, #12G10), rabbit polyclonal anti-phospho-Akt (Ser473) antibodies (Cell Signaling Technology, #9271), rabbit polyclonal anti-Akt antibodies (Cell Signaling Technology, #9272), and mouse monoclonal anti-FLAG antibodies.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Small GTPase Rab17 Regulates the Surface Expression of Kainate Receptors but Not α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors in Hippocampal Neurons via Dendritic Trafficking of Syntaxin-4 Protein *

doi: 10.1074/jbc.M114.550632

Figure Lengend Snippet: Rab17 co-localizes with Syntaxin-4 in the soma. A , representative images of plasma membrane-associated Syntaxins in developing neurons. At 8 DIV, mouse hippocampal neurons were transfected with pCMV-Myc-Stx1 ( left panel ), pCMV-Myc-Stx2 ( 2nd panel from the left ), pCMV-Myc-Stx3 ( 3rd panel from the left ), or pCMV-Myc-Stx4 ( right panel ), and at 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Myc ( black ) and MAP2 ( red ). The arrows and arrowheads point to axons and dendrites, respectively. The lower four panels a–d are magnified views of the boxed areas in the top right panels. Bar, 10 μm. B, representative images of Rab17 and plasma membrane-associated Syntaxins at the soma in developing neurons. At 8 DIV, mouse hippocampal neurons were transfected with pEGFP-Rab17 together with pCMV-Myc-Stx1 ( top panel ), pCMV-Myc-Stx2 ( 2nd panel ), pCMV-Myc-Stx3 ( 3rd panel ), or pCMV-Myc-Stx4 ( bottom panel ), and at 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Myc ( red ) and MAP2. The dashed lines indicate dendritic shafts identified as MAP2-positive areas. Bar, 5 μm. C, quantification of the Pearson's correlation coefficient between EGFP-Rab17 and Myc-Syntaxin-1 ( n = 10), Myc-Syntaxin-2 ( n = 10), Myc-Syntaxin-3 ( n = 10), and Myc-Syntaxin-4 ( n = 10), as shown in A. **, p < 0.0025. D, representative images of endogenous Rab17 and Myc-Syntaxin-4 in the soma at developing neurons. At 8 DIV, hippocampal neurons were transfected with pCMV-Myc-Stx4, and at 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Rab17 ( green ), Myc ( red ), and MAP2. The dashed lines indicate MAP2-positive areas, and the arrows indicate the co-localization points. Bar, 5 μm.

Article Snippet: Commercially obtained antibodies were as follows: anti-c-Myc (9E10) mouse monoclonal antibody (Santa Cruz Biotechnology); anti-calcium/calmodulin-dependent protein kinase II α (6G9) mouse monoclonal antibody, anti-GAPDH (6C5) mouse monoclonal antibody, and anti-MAP2 chick polyclonal antibody (Millipore Corp.); anti-GFP (clones 7.1 and 13.1) mouse monoclonal antibody (Roche Applied Science); anti-Syntaxin-4 rabbit polyclonal antibody (Synaptic Systems); IRDye®680RD/800CW-conjugated anti-mouse/rabbit IgG donkey antibody (LI-COR Biotechnology); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Sigma); and Cy2/Cy3/Cy5 or Alexa Fluor® 488/594/647-conjugated anti-mouse/rabbit/chick/sheep IgG donkey antibody (Jackson ImmunoResearch).

Techniques: Transfection, Immunocytochemistry

Journal: The Journal of Biological Chemistry

Article Title: Small GTPase Rab17 Regulates the Surface Expression of Kainate Receptors but Not α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors in Hippocampal Neurons via Dendritic Trafficking of Syntaxin-4 Protein *

doi: 10.1074/jbc.M114.550632

Figure Lengend Snippet: Rab17 co-localizes with Syntaxin-4 in the dendritic shaft, tip, and spine. A, representative images of EGFP-Rab17 and Myc-Syntaxin-4 in the dendrite at developing neurons. At 8 DIV, mouse hippocampal neurons were co-transfected with pEGFP-Rab17 and pCMV-Myc-Stx4, and at 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Myc ( red ) and MAP2. The bottom panels are magnified views of the boxed area in the top right panel , and the dashed lines indicate dendritic shafts identified as MAP2-positive areas. The arrows indicate the co-localization points. Bar, 10 μm. B, representative images of Rab17 and plasma membrane-associated Syntaxins in the distal dendrite at developing neurons. At 8 DIV, mouse hippocampal neurons were transfected with pCMV-Myc-Stx1 ( top panels ), pCMV-Myc-Stx2 ( 2nd panel ), pCMV-Myc-Stx3 ( 3rd panel ), or pCMV-Myc-Stx4 ( bottom panel ), and at 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Rab17 ( green ), Myc ( red ), and MAP2. The dashed lines indicate dendritic shafts identified as MAP2-positive areas. The arrows indicate the co-localization points. Bar, 5 μm. C, representative images of Rab17 and plasma membrane-associated Syntaxins in the spine at matured neurons. At 8 DIV, mouse hippocampal neurons were transfected with pCMV-Myc-Stx1 ( top panels ), pCMV-Myc-Stx2 ( 2nd panel ), pCMV-Myc-Stx3 ( 3rd panel ), or pCMV-Myc-Stx4 ( bottom panel ), and at 21 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Rab17 ( green ), Myc ( red ), and MAP2. The dashed lines indicate dendritic shafts identified as MAP2-positive areas. The arrows indicate the co-localization points. Bar, 5 μm. D, representative images of Rab17 and Myc-Syntaxin-4 in the mature neurons. At 8 DIV, rat hippocampal neurons were transfected with pCMV-Myc-Stx4, and at 24 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Rab17 ( green ), Myc ( red ), and calcium/calmodulin-dependent protein kinase II α ( CaMKII α) (a spine marker; blue ). Bar, 5 μm. The arrows indicate the co-localization points.

Article Snippet: Commercially obtained antibodies were as follows: anti-c-Myc (9E10) mouse monoclonal antibody (Santa Cruz Biotechnology); anti-calcium/calmodulin-dependent protein kinase II α (6G9) mouse monoclonal antibody, anti-GAPDH (6C5) mouse monoclonal antibody, and anti-MAP2 chick polyclonal antibody (Millipore Corp.); anti-GFP (clones 7.1 and 13.1) mouse monoclonal antibody (Roche Applied Science); anti-Syntaxin-4 rabbit polyclonal antibody (Synaptic Systems); IRDye®680RD/800CW-conjugated anti-mouse/rabbit IgG donkey antibody (LI-COR Biotechnology); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Sigma); and Cy2/Cy3/Cy5 or Alexa Fluor® 488/594/647-conjugated anti-mouse/rabbit/chick/sheep IgG donkey antibody (Jackson ImmunoResearch).

Techniques: Transfection, Immunocytochemistry, Marker

Journal: The Journal of Biological Chemistry

Article Title: Small GTPase Rab17 Regulates the Surface Expression of Kainate Receptors but Not α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors in Hippocampal Neurons via Dendritic Trafficking of Syntaxin-4 Protein *

doi: 10.1074/jbc.M114.550632

Figure Lengend Snippet: Rab17 is required for polarized trafficking of Syntaxin-4 to the dendrite. A–D, representative images of plasma membrane-associated Syntaxins in the Rab17 -shRNA-transfected neurons. At 8 DIV, mouse hippocampal neurons were transfected with pCMV-Myc-Stx1 ( A ), pCMV-Myc-Stx2 ( B ), pCMV-Myc-Stx3 ( C ), or pCMV-Myc-Stx4 ( D ) together with pSilencer-CMV-EGFP-Control or pSilencer-CMV-EGFP-shRab17, and at 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Myc ( black ), GFP ( green ), and MAP2 ( red ). The arrows and arrowheads point to axons and dendrites, respectively. The lower three panels a–c are magnified views of the boxed areas in the top right panels. Bar, 10 μm. E, quantification of the proportion of Myc-Syntaxin-1 ( n = 10), Myc-Syntaxin-2 ( n = 10), Myc-Syntaxin-3 ( n = 10), and Myc-Syntaxin-4 ( n = 10) in the control neurons and Rab17 -shRNA-transfected neurons at the dendrites, as shown in A–D . The rate of translocated level of Myc-Syntaxin-4 was calculated by dividing the dendrite Myc-Syntaxin-4 fluorescence intensity by the total Myc-Syntaxin-4 fluorescence intensity. **, p < 0.0025. F, quantification of the fluorescence ratio of Myc-Syntaxin-4 between dendrite and axon in the control neurons and Rab17 -shRNA transfected neurons, as shown in D . **, p < 0.0025.

Article Snippet: Commercially obtained antibodies were as follows: anti-c-Myc (9E10) mouse monoclonal antibody (Santa Cruz Biotechnology); anti-calcium/calmodulin-dependent protein kinase II α (6G9) mouse monoclonal antibody, anti-GAPDH (6C5) mouse monoclonal antibody, and anti-MAP2 chick polyclonal antibody (Millipore Corp.); anti-GFP (clones 7.1 and 13.1) mouse monoclonal antibody (Roche Applied Science); anti-Syntaxin-4 rabbit polyclonal antibody (Synaptic Systems); IRDye®680RD/800CW-conjugated anti-mouse/rabbit IgG donkey antibody (LI-COR Biotechnology); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Sigma); and Cy2/Cy3/Cy5 or Alexa Fluor® 488/594/647-conjugated anti-mouse/rabbit/chick/sheep IgG donkey antibody (Jackson ImmunoResearch).

Techniques: shRNA, Transfection, Immunocytochemistry, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Small GTPase Rab17 Regulates the Surface Expression of Kainate Receptors but Not α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors in Hippocampal Neurons via Dendritic Trafficking of Syntaxin-4 Protein *

doi: 10.1074/jbc.M114.550632

Figure Lengend Snippet: Rab17 is required for dendritic trafficking of endogenous Syntaxin-4. A, HEK293T cells were transfected with pFIV-Control ( lanes 1, 4, and 6 ), pFIV-shStx4-1 ( lanes 2, 5, and 7 ), or pFIV-shStx4-2 ( lane 3 ) together with pCMV-Myc-ratStx4 ( lanes 1–3 ), pCMV-Myc-Stx4 SR ( lanes 4 and 5 ), or pCMV-Myc-Stx-3 ( lanes 6 and 7 ). Two days after transfection, the cells were lysed and subjected to immunoblot analysis with anti-Myc antibody ( upper panel ) and anti-GAPDH antibody ( lower panel ). B, Neuro2A cells were transfected with pFIV-Control ( lane 1 ), pFIV-shStx4-1 ( lane 2 ), or pFIV-shStx4-2 ( lane 3 ). Two days after transfection the cells were lysed and subjected to immunoblot analysis with anti-Syntaxin-4 antibody ( upper panel ) and anti-GAPDH antibody ( lower panel ). C, quantification of endogenous Syntaxin-4 of control-shRNA, Stx4 -shRNA-1 and Stx4 -shRNA-2 as shown in B. A.U. , arbitrary units. **, p < 0.0025. D, representative images of Myc-Syntaxin-4-expressing neurons in the presence and absence of Stx4 -shRNA. At 8 DIV, rat hippocampal neurons were transfected with pCMV-Myc-ratStx4 together with pFIV-Control, pFIV-shStx4-1, or pFIV-shStx4-2, and at 11 DIV, the neurons were fixed. The neurons were stained by Myc ( red ). Bar, 10 μm. E, quantification of the Myc-Syntaxin-4 of control-shRNA-transfected neurons ( n = 10), Stx4 -shRNA-1-transfected neurons ( n = 10), and Stx4 -shRNA-2-transfected neurons ( n = 10) as shown in B. A.U. , arbitrary units. **, p < 0.0025. F, representative images of Myc-Syntaxin-4-expressing neurons stained by Syntaxin-4 antibody. At 8 DIV, rat hippocampal neurons were transfected with pCMV-Myc-Stx4, and at 11 DIV, the neurons were fixed. The neurons were stained by Myc ( green ), Syntaxin-4 ( black ), and MAP2 ( red ). Bar, 10 μm. G, representative images of endogenous Syntaxin-4 in the presence and absence of Stx4 -shRNA. At 8 DIV, rat hippocampal neurons were transfected with pFIV-Control and pFIV-shStx4-1, and at 11 DIV, the neurons were fixed. The neurons were stained by Syntaxin-4 ( black ) and MAP2 ( red ). Bar, 10 μm. H, quantification of endogenous Syntaxin-4 of control-shRNA-transfected neurons ( n = 10) and Stx4 -shRNA-1-transfected neurons ( n = 10) shown in G. A.U., arbitrary units. **, p < 0.0025. I, representative images of endogenous Syntaxin-4 in the Rab17 -shRNA-transfected neurons. At 8 DIV, mouse hippocampal neurons were transfected with pSilencer-CMV-EGFP-Control or pSilencer-CMV-EGFP-shRab17, and at 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Syntaxin-4 ( black ), GFP ( green ), and MAP2 ( red ). The arrows and arrowheads point to axons and dendrites, respectively. The lower three panels a–c are magnified views of the boxed areas in the top right panels. Bar, 10 μm. J, quantification of the proportion of endogenous Syntaxin-4 in the control neurons ( n = 10) and Rab17 -shRNA-transfected neurons ( n = 10) at the dendrites, shown in I . The rate of translocated level of Syntaxin-4 was calculated by dividing the dendrite Syntaxin-4 fluorescence intensity by the total Syntaxin-4 fluorescence intensity. *, p < 0.025.

Article Snippet: Commercially obtained antibodies were as follows: anti-c-Myc (9E10) mouse monoclonal antibody (Santa Cruz Biotechnology); anti-calcium/calmodulin-dependent protein kinase II α (6G9) mouse monoclonal antibody, anti-GAPDH (6C5) mouse monoclonal antibody, and anti-MAP2 chick polyclonal antibody (Millipore Corp.); anti-GFP (clones 7.1 and 13.1) mouse monoclonal antibody (Roche Applied Science); anti-Syntaxin-4 rabbit polyclonal antibody (Synaptic Systems); IRDye®680RD/800CW-conjugated anti-mouse/rabbit IgG donkey antibody (LI-COR Biotechnology); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Sigma); and Cy2/Cy3/Cy5 or Alexa Fluor® 488/594/647-conjugated anti-mouse/rabbit/chick/sheep IgG donkey antibody (Jackson ImmunoResearch).

Techniques: Transfection, Western Blot, shRNA, Expressing, Staining, Immunocytochemistry, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Small GTPase Rab17 Regulates the Surface Expression of Kainate Receptors but Not α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors in Hippocampal Neurons via Dendritic Trafficking of Syntaxin-4 Protein *

doi: 10.1074/jbc.M114.550632

Figure Lengend Snippet: Syntaxin-4 is necessary for the surface expression of GluK2. A, representative images of surface expression level of Myc-GluK2 in the Syntaxin-4 -shRNA-transfected neurons. At 8 DIV, rat hippocampal neurons were transfected with pCDNA3-Myc-GluK2 and pEGFP-C2 together with pFIV-Control or pFIV-shStx4-1, and at 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Myc (surface, red and total, green ). GFP image was replaced black in this figure. Bar, 10 μm. B, quantification of the surface expression level of Myc-GluK2 of control-shRNA-transfected neurons as indicated ( n = 10 for all conditions). At 8 DIV, rat hippocampal neurons were transfected with pCDNA3-Myc-GluK2 and pEGFP-C2 together with pFIV-Control, pFIV-shStx4-1, pFIV-shStx4-2, pFIV-shRab17, pFIV-shStx4-1, and pFIV-shRab17, or pFIV-shStx4-1 and pCMV-Stx4 SR , and at 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Myc (surface, red and total, green ). Surface expression level of Myc-GluK2 was calculated by dividing the surface Myc-GluK2 fluorescence intensity by the total Myc-GluA1 fluorescence intensity. A.U. , arbitrary units. **, p < 0.0025; *, p < 0.025. N.S. , not significant. C, representative images of surface expression level of Myc-GluA1 in the Syntaxin-4 -shRNA-transfected neurons. At 8 DIV, rat hippocampal neurons were transfected with pCDNA3-Myc-GluA1 and pEGFP-C2 together with pFIV-Control or pFIV-shStx4-1, and at 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Myc (surface red and total, green ). GFP image was replaced black in this figure. D, quantification of the surface expression level of Myc-GluA1 of control-shRNA-transfected neurons ( n = 10) and Stx4 -shRNA-1-transfected neurons ( n = 10) as shown in C . Surface expression level of Myc-GluA1 was calculated by dividing the surface Myc-GluA1 fluorescence intensity by the total Myc-GluA1 fluorescence intensity. A.U. , arbitrary units. N.S. , not significant.

Article Snippet: Commercially obtained antibodies were as follows: anti-c-Myc (9E10) mouse monoclonal antibody (Santa Cruz Biotechnology); anti-calcium/calmodulin-dependent protein kinase II α (6G9) mouse monoclonal antibody, anti-GAPDH (6C5) mouse monoclonal antibody, and anti-MAP2 chick polyclonal antibody (Millipore Corp.); anti-GFP (clones 7.1 and 13.1) mouse monoclonal antibody (Roche Applied Science); anti-Syntaxin-4 rabbit polyclonal antibody (Synaptic Systems); IRDye®680RD/800CW-conjugated anti-mouse/rabbit IgG donkey antibody (LI-COR Biotechnology); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Sigma); and Cy2/Cy3/Cy5 or Alexa Fluor® 488/594/647-conjugated anti-mouse/rabbit/chick/sheep IgG donkey antibody (Jackson ImmunoResearch).

Techniques: Expressing, shRNA, Transfection, Immunocytochemistry, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Small GTPase Rab17 Regulates the Surface Expression of Kainate Receptors but Not α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors in Hippocampal Neurons via Dendritic Trafficking of Syntaxin-4 Protein *

doi: 10.1074/jbc.M114.550632

Figure Lengend Snippet: Active form of Rab17 promotes surface expression of GluK2 by enhancing Syntaxin-4 translocation to dendrites. A, representative images of Myc-Syntaxin-4 in active form Rab17-expressing neurons. At 8 DIV, rat hippocampal neurons were transfected with pCMV-Myc-Stx4 together with pEGFP-C2 or pEGFP-Rab17-Q77L. At 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Myc ( black ) and MAP2 ( green ). The bottom three panels a–c are magnified views of the boxed areas in the top right panels. Bar, 10 μm. B, quantification of the proportion of Myc-Syntaxin-4 in the dendrites in the presence of EGFP ( n = 10) and EGFP-Rab17-Q77L as shown in A . The rate of translocated level of Myc-Syntaxin-4 was calculated by dividing the dendrite Myc-Syntaxin-4 fluorescence intensity by the total Myc-Syntaxin-4 fluorescence intensity. **, p < 0.0025. C , representative images of surface expression level of SEP-GluK2 in active form Rab17 and Stx4 -shRNA-transfected neurons. At 8 DIV, rat hippocampal neurons were transfected with pCDNA3-SEP-Myc-GluK2 together with pCAG-Control and pFIV-Control, pCAG-Control, and pFIV-shStx4-1, pCAG-Myc-Rab17-Q77L, and pFIV-Control, or pCAG-Myc-Rab17-Q77L and pFIV-shStx4-1. At 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against GFP (surface, red and total, green ) and Rab17 ( black ). Bar, 10 μm. D, quantification of the surface expression level of SEP-GluK2 of control neurons ( n = 10), Stx4 -shRNA-1-transfected neurons ( n = 10), Rab17-Q77L-transfected neurons ( n = 10), and Rab17-Q77L- and Stx4 -shRNA-1-transfected neurons ( n = 10) as shown in C . Surface expression level of SEP-GluK2 was calculated by dividing the surface SEP-GluK2 fluorescence intensity by the total SEP-GluK2 fluorescence intensity. A.U., arbitrary units. *, p < 0.025; **, p < 0.0025.

Article Snippet: Commercially obtained antibodies were as follows: anti-c-Myc (9E10) mouse monoclonal antibody (Santa Cruz Biotechnology); anti-calcium/calmodulin-dependent protein kinase II α (6G9) mouse monoclonal antibody, anti-GAPDH (6C5) mouse monoclonal antibody, and anti-MAP2 chick polyclonal antibody (Millipore Corp.); anti-GFP (clones 7.1 and 13.1) mouse monoclonal antibody (Roche Applied Science); anti-Syntaxin-4 rabbit polyclonal antibody (Synaptic Systems); IRDye®680RD/800CW-conjugated anti-mouse/rabbit IgG donkey antibody (LI-COR Biotechnology); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Sigma); and Cy2/Cy3/Cy5 or Alexa Fluor® 488/594/647-conjugated anti-mouse/rabbit/chick/sheep IgG donkey antibody (Jackson ImmunoResearch).

Techniques: Expressing, Translocation Assay, Transfection, Immunocytochemistry, Fluorescence, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Small GTPase Rab17 Regulates the Surface Expression of Kainate Receptors but Not α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors in Hippocampal Neurons via Dendritic Trafficking of Syntaxin-4 Protein *

doi: 10.1074/jbc.M114.550632

Figure Lengend Snippet: Syntaxin-4 is not required for the control of dendritic morphogenesis. A, typical images of Rab17- and Syntaxin4-knockdown neurons. At 8 DIV, hippocampal neurons were transfected with a vector encoding EGFP and control-shRNA ( upper panel ), Rab17 -shRNA ( middle panel ), or Stx4 -shRNA-1 ( lower panel ), and at 11 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against MAP2 ( red ). The arrows and arrowheads point to axons and dendrites, respectively. Bar, 50 μm. B and C, quantification of the total dendrite length ( B ) and total dendrite branching tip numbers ( C ) of the control neurons ( n = 20), Rab17-knockdown neurons ( n = 20), and Syntaxin-4-knockdown neurons ( n = 20). **, p < 0.0025. N.S. , not significant.

Article Snippet: Commercially obtained antibodies were as follows: anti-c-Myc (9E10) mouse monoclonal antibody (Santa Cruz Biotechnology); anti-calcium/calmodulin-dependent protein kinase II α (6G9) mouse monoclonal antibody, anti-GAPDH (6C5) mouse monoclonal antibody, and anti-MAP2 chick polyclonal antibody (Millipore Corp.); anti-GFP (clones 7.1 and 13.1) mouse monoclonal antibody (Roche Applied Science); anti-Syntaxin-4 rabbit polyclonal antibody (Synaptic Systems); IRDye®680RD/800CW-conjugated anti-mouse/rabbit IgG donkey antibody (LI-COR Biotechnology); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Sigma); and Cy2/Cy3/Cy5 or Alexa Fluor® 488/594/647-conjugated anti-mouse/rabbit/chick/sheep IgG donkey antibody (Jackson ImmunoResearch).

Techniques: Transfection, Plasmid Preparation, shRNA, Immunocytochemistry

Journal: The Journal of Biological Chemistry

Article Title: Small GTPase Rab17 Regulates the Surface Expression of Kainate Receptors but Not α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors in Hippocampal Neurons via Dendritic Trafficking of Syntaxin-4 Protein *

doi: 10.1074/jbc.M114.550632

Figure Lengend Snippet: Schematic representation of proposed roles of Rab17. Rab17 mediates polarized trafficking of Syntaxin-4, which controls GluK2 but not GluA1 surface insertion at the dendrite in hippocampal neurons.

Article Snippet: Commercially obtained antibodies were as follows: anti-c-Myc (9E10) mouse monoclonal antibody (Santa Cruz Biotechnology); anti-calcium/calmodulin-dependent protein kinase II α (6G9) mouse monoclonal antibody, anti-GAPDH (6C5) mouse monoclonal antibody, and anti-MAP2 chick polyclonal antibody (Millipore Corp.); anti-GFP (clones 7.1 and 13.1) mouse monoclonal antibody (Roche Applied Science); anti-Syntaxin-4 rabbit polyclonal antibody (Synaptic Systems); IRDye®680RD/800CW-conjugated anti-mouse/rabbit IgG donkey antibody (LI-COR Biotechnology); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Sigma); and Cy2/Cy3/Cy5 or Alexa Fluor® 488/594/647-conjugated anti-mouse/rabbit/chick/sheep IgG donkey antibody (Jackson ImmunoResearch).

Techniques: