Journal: Journal of pharmacological sciences

Article Title: Syntaxin 3 interacts with serotonin transporter and regulates its function.

doi: 10.1016/j.jphs.2021.01.007

Figure Lengend Snippet: Fig. 1. Interaction of SERT with STX3 in COS-7 cells overexpressing SERT and STX3. A: Immunoprecipitation studies using COS-7 cells overexpressing SERT and STX3. COS-7 cells were transfected with FLAG-SERT and untagged STX3 cDNA or mock vector (pcDNA3.0). Cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG-tagged mAb magnetic agarose beads and analyzed by immunoblotting (IB) with an anti-FLAG antibody or anti-STX3 antibody. The cell lysate inputs used for IP were blotted with anti-FLAG or anti-STX3 antibodies. Immunoprecipitated samples prepared from cells expressing both STX3 and FLAG-SERT contained STX3 (black circles). Arrowheads and arrows indicate mature glycosylated SERT and immaturely glycosylated SERT, respectively. An asterisk indicates a nonspecific band. The figure shows the representative data from two independent experiments. B: Immunoprecipitation studies using normal mouse IgG and anti-FLAG antibodies. COS-7 cells were transfected with FLAG-SERT and untagged STX3 cDNA. Cell lysates were subjected to IP with normal mouse IgG, followed by protein G magnetic sepharose beads or anti-FLAG-tagged mAb magnetic agarose beads, and analyzed by IB with an anti-FLAG antibody or anti-STX3 antibody. The immunoprecipitated samples that reacted with an anti-FLAG antibody contain STX3 (black circle), while those that reacted with normal IgG do not contain STX3. An asterisk indicates a nonspecific band. SERT, serotonin transporter; STX$, syntaxin-3.

Article Snippet: To generate STX3-overexpressing cells, plasmids expressing Myc-DDK-tagged human STX3 (pMyc-DDK-STX3) and untagged human STX3 (pSTX3) were purchased from Origene (Rockville, MD, USA).

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Journal of pharmacological sciences

Article Title: Syntaxin 3 interacts with serotonin transporter and regulates its function.

doi: 10.1016/j.jphs.2021.01.007

Figure Lengend Snippet: Fig. 2. Localization of SERT and STX3 in COS-7 cells. A: Immunocytochemical anal- ysis of COS-7 cells transfected with pHA-SERT and pMyc-DDK-STX3. Forty-eight hours after transfection, immunocytochemical studies using an anti-HA antibody or anti-Myc antibody were performed. HA-SERT and Myc-DDK-STX3 are colocalized in some intracellular organelles of COS-7 cells as aggregate-like structures. Bar: 10 mm. B: Comparison of SERT and STX3 localization in the endoplasmic reticulum (ER), as visualized with KDEL antibody, in COS-7 cells. HA-SERT is localized in the ER, which was labeled with an anti-KDEL antibody (upper panel). Myc-DDK-STX3 is also localized in the ER (lower panel). Bar: 10 mm. C: Comparison of SERT and STX3 localization with the Golgi apparatus, as visualized by an anti-GM130 antibody, in COS-7 cells. HA-SERT is localized in the Golgi apparatus, which was labeled with an anti-GM130 antibody (upper panel). Myc-DDK-STX3 was also localized in the Golgi apparatus (lower panel). Bar: 10 mm. SERT, serotonin transporter; STX$, syntaxin-3.

Article Snippet: To generate STX3-overexpressing cells, plasmids expressing Myc-DDK-tagged human STX3 (pMyc-DDK-STX3) and untagged human STX3 (pSTX3) were purchased from Origene (Rockville, MD, USA).

Techniques: Transfection, Comparison, Labeling

Journal: Journal of pharmacological sciences

Article Title: Syntaxin 3 interacts with serotonin transporter and regulates its function.

doi: 10.1016/j.jphs.2021.01.007

Figure Lengend Snippet: Fig. 3. Effects of STX3 overexpression on the uptake activity, protein expression, and glycosylation of SERT. A: Effects of STX3 overexpression on SERT uptake activity. In cells expressing FLAG-SERT and mock (pcDNA) or Myc-DDK-STX3, we measured the uptake of fluorescent SERT substrate per cell as the 5-HT uptake activity. Uptake activity was evaluated using the Neurotransmitter Transporter Uptake Assay kit as described in the Materials and Methods section. STX3 overexpression significantly reduces the 5-HT uptake activity of SERT in AD293 and COS-7 cells [AD293 cells: *p < 0.0001, n ¼ 9 (three independent experiments), Student's t-test, compared to mock control; COS-7 cells: *p < 0.0001, n ¼ 12 (4 independent experiments), Student's t-test, compared to mock control]. B: Effects of STX3 overexpression on SERT protein expression and glycosylation. Representative immunoblotting data of FLAG-SERT expression in AD293, FLAG-SERT HEK, and COS-7 cells are shown. According to our previous studies, the band with a molecular size of approximately 80 kDa (arrowhead) is considered to be functional and maturely glycosylated SERT expressed at the plasma membrane, and the band with a molecular size of approximately 63 kDa (arrow) is considered to be high-mannose-type immaturely glycosylated SERT in the endoplasmic reticulum (ER). Furthermore, a band larger than 100 kDa is considered to be a dimer of immaturely glycosylated SERT.18,19,37 STX3 overexpression tends to decrease the expression of mature SERT in all cell types. C: Comparison of levels of maturely glycosylated SERT between mock-transfected and STX3-overexpressing COS-7 cells. There are no statistically significant differences between the groups, although STX3 overexpression tends to decrease the expression of mature SERT (n ¼ 5, ns). SERT, serotonin transporter; STX$, syntaxin-3; 5-HT, 5-hydroxytryptamine.

Article Snippet: To generate STX3-overexpressing cells, plasmids expressing Myc-DDK-tagged human STX3 (pMyc-DDK-STX3) and untagged human STX3 (pSTX3) were purchased from Origene (Rockville, MD, USA).

Techniques: Over Expression, Activity Assay, Expressing, Glycoproteomics, Control, Western Blot, Functional Assay, Clinical Proteomics, Membrane, Comparison, Transfection

Journal: Journal of pharmacological sciences

Article Title: Syntaxin 3 interacts with serotonin transporter and regulates its function.

doi: 10.1016/j.jphs.2021.01.007

Figure Lengend Snippet: Fig. 4. Effects of STX3 overexpression on SERT membrane expression. A: Comparison of the membrane expression of HA-SERT in Myc-STX3-overexpressing and non-Myc-STX3- overexpressing cells. Immunofluorescence staining of COS-7 cells transfected with pHA-SERT and pMyc-DDK-STX3 using anti-HA and anti-Myc antibodies, respectively, was performed. Fluorescence signals were observed using a confocal laser scanning microscope. HA-SERT appears to be expressed throuout Myc-STX3-expressing cells (arrows). In contrast, HA-SERT is preferentially expressed at the plasma membrane in non-Myc-STX3-expressing cells (arrowhead). Bar: 10 mm. B: Comparison of SERT membrane expression between STX3-overexpressing and non-STX3-expressing cells using line profiling of SERT immunofluorescence. Line profile analysis was performed along the white lines, which did not cross the nucleus. Bar: 10 mm. C: Representative line profile data from STX3-overexpressing and non-STX3-expressing cells. In non-STX3-expressing cells, the SERT fluorescence intensity is high in the cell membrane, whereas the fluorescence intensity was spread evenly in STX3-expressing cells. Red lines indicate the peak fluorescence intensity corre- sponding to the plasma membrane expression of SERT. The green line indicates the scanning distance of the line profiling. D: Quantitative analysis of SERT membrane expression using line profiling analysis. SERT membrane expression was quantitatively analyzed as described in the Materials and Methods section. Overexpression of STX3 significantly decreases the membrane expression of SERT (*p < 0.001, Student's t-test, compared with non-STX3-expressing control cells, n ¼ 39 in the non-STX3-expressing group, n ¼ 33 in the STX3-overexpressing group). Three independent experiments were conducted. SERT, serotonin transporter; STX$, syntaxin-3.

Article Snippet: To generate STX3-overexpressing cells, plasmids expressing Myc-DDK-tagged human STX3 (pMyc-DDK-STX3) and untagged human STX3 (pSTX3) were purchased from Origene (Rockville, MD, USA).

Techniques: Over Expression, Membrane, Expressing, Comparison, Staining, Transfection, Fluorescence, Laser-Scanning Microscopy, Clinical Proteomics, Control

Journal: Journal of pharmacological sciences

Article Title: Syntaxin 3 interacts with serotonin transporter and regulates its function.

doi: 10.1016/j.jphs.2021.01.007

Figure Lengend Snippet: Fig. 5. Effects of STX3 knockdown on SERT uptake activity and the levels of SERT. A: Confirmation of STX3 knockdown in AD293 cells by transfection with STX3 siRNA. AD293 cells were transfected with STX3 siRNA or control siRNA concomitantly with FLAG-SERT. Immunoblotting using an anti-STX3 antibody confirmed that STX3 levels are reduced by STX3 siRNA. Similar results were obtained when COS-7 or FLAG-SERT HEK cells were used (data not shown). B: Effects of STX3 knockdown on SERT uptake activity in AD293, COS-7, and FLAG-SERT HEK cells. STX3 siRNA or control siRNA was transfected into AD293, COS-7, and FLAG-SERT HEK cells concomitantly with pFLAG-SERT. We measured the uptake of fluorescent SERT substrate per cell as the 5-HT uptake activity, as described in the Materials and Methods section. Knockdown of STX3 does not demonstrate any significant effect on SERT uptake activity in any cell type (AD293 cells: n ¼ 9, 3 independent experiments, ns, Student's t-test; FLAG-SERT HEK cells: n ¼ 9, 3 independent experiments, ns, Student's t- test; COS-7 cells: n ¼ 18, 6 independent experiments, ns, Student's t-test). C: Effects of STX3 knockdown on the protein level of SERT expressed in COS-7 cells. COS-7 cells were transfected with siRNAs and pFLAG-SERT. Immunoblotting using an anti-FLAG antibody shows that STX3 knockdown does not alter the protein levels of maturely glycosylated or immaturely glycosylated SERT (ns, n ¼ 5). SERT, serotonin transporter; STX$, syntaxin-3; 5-HT, 5-hydroxytryptamine.

Article Snippet: To generate STX3-overexpressing cells, plasmids expressing Myc-DDK-tagged human STX3 (pMyc-DDK-STX3) and untagged human STX3 (pSTX3) were purchased from Origene (Rockville, MD, USA).

Techniques: Knockdown, Activity Assay, Transfection, Control, Western Blot

Journal: Journal of pharmacological sciences

Article Title: Syntaxin 3 interacts with serotonin transporter and regulates its function.

doi: 10.1016/j.jphs.2021.01.007

Figure Lengend Snippet: Fig. 6. Effects of STX3 knockdown on SERT glycosylation. A: Transfection with STX3 siRNA causes the maturely glycosylated SERT band to shift upward. The maturely glycosylated SERT band (arrowhead) migrates slightly upward in STX3 siRNA-transfected AD293, FLAG-SERT-HEK, and COS-7 cells. Immaturely glycosylated SERT is barely observed in AD293 and FLAG-SERT HEK cells. The figure shows representative data from three to five independent experiments. B: Effects of the glycolytic enzymes PNGaseF and EndoH on the shift of the maturely glycosylated SERT band. Lysates of AD293 cells transfected with siRNAs and pFLAG-SERT were treated with the glycolytic enzyme PNGaseF or EndoH for 2 h at 37 C and were subjected to Western blot analysis. Treatment with PNGase F shifts the band of the maturely glycosylated SERT to a molecular size of 50 kDa (asterisk). No band shift is observed in the PNGase F-treated samples (asterisk). Conversely, Endo H does not affect the size of the maturely glycosylated SERT band (triangle). The figure shows the repre- sentative data from two independent experiments. SERT, serotonin transporter; STX$, syntaxin-3; 5-HT, 5-hydroxytryptamine.

Article Snippet: To generate STX3-overexpressing cells, plasmids expressing Myc-DDK-tagged human STX3 (pMyc-DDK-STX3) and untagged human STX3 (pSTX3) were purchased from Origene (Rockville, MD, USA).

Techniques: Knockdown, Glycoproteomics, Transfection, Western Blot, Electrophoretic Mobility Shift Assay

Journal: Journal of pharmacological sciences

Article Title: Syntaxin 3 interacts with serotonin transporter and regulates its function.

doi: 10.1016/j.jphs.2021.01.007

Figure Lengend Snippet: Fig. 7. Confirmation of SERT mRNA expression, 5-HT uptake activity, and localization of SERT and STX3 in Caco-2 cells. A: SERT mRNA expression in various cell lines. The mRNA expression of SERT in Caco-2, COS-7, and AD293 cells was investigated by real-time PCR. The expression of SERT mRNA in Caco-2 cells is high, whereas it is extremely low in COS-7 and AD293 cells. B: 5-HT uptake activity in Caco-2 cells. Caco-2 cells seeded in 24-well plates were subjected to the [3H] 5-HT uptake assay. The left column shows 5-HT uptake activity in the absence of 20 mM fluvoxamine, an SSRI. The right column shows 5-HT uptake activity in the presence of SSRI. The results reveal the existence of 5-HT uptake activity, which is probably mediated via SSRI-sensitive endogenous SERT in Caco-2 cells (n ¼ 4). C: Comparison of colocalization of SERT and STX3 with villin, a micro- villus marker, in Caco-2 cells. Caco-2 cells were immunostained with anti-SERT or anti-STX3 antibodies. The cells were then stained with an antibody against villin, a microvillus marker. SERT and STX3 are localized in punctate structures, which almost coincides with the localization of villin. The inset presents a magnified view of the indicated area, showing that STX3 is partially colocalized with villin (lower panel). Bar: 20 mm. D: Orthographic view of X- and Y-axis cross-sections constructed from a 3D image of villin and actin. Caco- 2 cells were immunostained with an anti-villin antibody and then with phalloidin-TRITC to stain actin and visualize the cell border. Villin staining reveals microvilli-like structures at the apical membrane (arrowhead). Red signals indicate actin, and green signals indicate villin. Bar: 20 mm. E: Comparison of orthographic views of SERT and STX3 with an orthographic view of the villin. Caco-2 cells were immunostained with anti-SERT or anti-STX3 antibodies. The cells were then stained with phalloidin-TRITC. Orthographic views of SERT and STX3 staining demonstrate that like villin (arrowhead), both SERT and STX are localized in microvilli-like structures. Red signals indicate actin, and green signals indicate SERT or STX3. Bar: 20 mm. SERT, serotonin transporter; STX$, syntaxin-3; 5-HT, 5-hydroxytryptamine; SSRI, selective serotonin reuptake inhibitor.

Article Snippet: To generate STX3-overexpressing cells, plasmids expressing Myc-DDK-tagged human STX3 (pMyc-DDK-STX3) and untagged human STX3 (pSTX3) were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Comparison, Marker, Staining, Construct, Membrane

Journal: Journal of pharmacological sciences

Article Title: Syntaxin 3 interacts with serotonin transporter and regulates its function.

doi: 10.1016/j.jphs.2021.01.007

Figure Lengend Snippet: Fig. 8. Effects of STX3 knockdown on 5-HT uptake in Caco-2 cells. A: Confirmation of STX3 knockdown in Caco-2 cells. Caco-2 cells were transfected with either the control or STX3 siRNA. Immunoblotting analysis using an anti-STX3 antibody was performed 48 h after transfection. Transfection with STX3 siRNA reduces the levels of STX3 in Caco-2 cells. B: Effects of STX3 knockdown on 5-HT uptake in Caco-2 cells. A [3H] 5-HT uptake assay was performed 48 h after transfection. Transfection with STX3 siRNA marginally but significantly reduces 5-HT uptake activity in Caco-2 cells (*p < 0.00.5 (Student's t-test, n ¼ 28, 7 independent experiments). SERT, serotonin transporter; STX$, syntaxin-3; 5-HT, 5- hydroxytryptamine.

Article Snippet: To generate STX3-overexpressing cells, plasmids expressing Myc-DDK-tagged human STX3 (pMyc-DDK-STX3) and untagged human STX3 (pSTX3) were purchased from Origene (Rockville, MD, USA).

Techniques: Knockdown, Transfection, Control, Western Blot, Activity Assay

Journal: iScience

Article Title: Legionella employs the multimodal ubiquitination of Sec22b to modulate SNARE pairing

doi: 10.1016/j.isci.2025.114341

Figure Lengend Snippet: Ubiquitination of Sec22b promotes non-canonical SNARE pairings with Stx3 and Stx4 (A) HEK293T-FcγRII cells stably expressing FLAG-Sec22b were infected with the indicated L. pneumophila strains at an MOI of 80 for 1 h. FLAG-Sec22b derivatives were immunoprecipitated from the cell lysates at the indicated time points using anti-FLAG beads. The cell lysates (input) and the immunoprecipitated proteins (IP: FLAG) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The intensity values of Stx3 protein bands were quantified using the ChemiDoc System with Image Lab software (Bio-Rad), normalized to the value of the no-infection condition (no bacteria), and displayed below the immunoblotting data. (B) HEK293T-FcγRII cells stably expressing FLAG-Sec22b S137A were infected with the indicated L. pneumophila strains at an MOI of 80 for 1 h, and analyzed as described in (A). The intensity values of Stx3 protein bands were quantified as described in (A) and displayed below the immunoblotting data. (C) HEK293T-FcγRII cells stably expressing FLAG-Sec22b or FLAG-Sec22b S137A were infected with the wild-type strain Lp01 at an MOI of 80 for 1 h, and analyzed as described in (A). (D) Intensity values of Stx3, Stx4, and Stx18 protein bands from (C) quantified as described in (A), normalized to the respective Syntaxin values bound to Sec22b WT. Values represent the mean ± SEM from three independent experiments. ns, not significant. ∗ p < 0.05, ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-Stx3 rabbit polyclonal antibody (15556-1-AP) and anti-Stx4 rabbit monoclonal antibody (14988-1-AP) were purchased from Proteintech.

Techniques: Ubiquitin Proteomics, Stable Transfection, Expressing, Infection, Immunoprecipitation, SDS Page, Western Blot, Software, Bacteria

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival

doi: 10.1073/pnas.2010751117

Figure Lengend Snippet: Conditional knockout of Stx3 eliminates STX3 expression in photoreceptors. (A) Retinal sections were immunofluorescently labeled for STX3 (red), Cre (green), and nuclei (blue). (B) STX3 protein levels in Stx3f/f(iCre75) (P30) and Stx3f/f(CRX-Cre) (P15) retinas were assessed and plotted. Values are mean ± SEM of n = 3 independent samples. *P < 0.05, **P < 0.01 by unpaired two-tailed Student’s t test. (C) Retinal sections were labeled for STX3 (red). (Scale bars: 10 µm in A and C.)

Article Snippet: Stx3 flox/flox ( Stx3 f/f ) mice were generated by inGenious Targeting Laboratory, Inc., Ronkonkoma, NY and crossed with iCre75 transgenic mice (generated by Ching-Kang Chen, Baylor College of Medicine, Houston TX and made available with permission by Wolfgang Baehr, University of Utah, Salt Lake City, UT) ( 26 ) or CRX-Cre transgenic mice (generated by Tom M. Glaser, University of California, Davis, CA and made available with permission by Anand Swaroop, National Eye Institute, NIH, Bethesda, MD).

Techniques: Knock-Out, Expressing, Labeling, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival

doi: 10.1073/pnas.2010751117

Figure Lengend Snippet: Elimination of STX3 leads to photoreceptor degeneration. (A) Representative light microscopic images. Red bars highlight OS thickness. (B) Nuclei in the ONL were counted along the vertical meridian of the eye from the superior to the inferior pole. Measurements were taken from three mice per genotype and time point and plotted as mean ± SD. + denotes comparisons between WT and Stx3f/f(iCre75) retina; * denotes comparisons between WT and Stx3f/f(CRX-Cre) retina. One symbol P < 0.05, two symbols P < 0.01, three symbols P < 0.001, four symbols P < 0.0001 in two-way ANOVA followed by Tukey’s post hoc comparisons. (C) INL thickness was measured in the inferior central (solid bars) and superior central retina (hashed bars). n = 3 mice per genotype, plotted as mean ± SD (D–J). TEM was from the indicated ages/genotypes. Arrows highlight IS abnormalities (D) and OS abnormalities (G). Insets are expanded views of regions in black boxes. (J) Additional TEM showing ISs and connecting cilia (arrows). Black outlines highlight cell borders around the IS/OS junction. (K) Retinal sections from adult (WT), P21 [Stx3f/f(iCre75)], and P15 [Stx3f/f(CRX-Cre)] were labeled for RPGR (green) and acetylated α-tubulin (red). (Right) Expanded views of white boxed regions. (L) Connecting cilia/axonemes were measured based on tubulin labeling from 100 photoreceptors from n = 3 mice per genotype for a total of 300 axonemes/genotype at the same ages as in K. Plotted is mean ± SD, ****P < 0.0001 in one-way ANOVA with Tukey’s post hoc comparison. (Scale bars: 20 µm in A; 2 µm in D, F, and H; 1 µm in E, G, I, and J; 10 µm in K; and 3 µm in K, Right.)

Article Snippet: Stx3 flox/flox ( Stx3 f/f ) mice were generated by inGenious Targeting Laboratory, Inc., Ronkonkoma, NY and crossed with iCre75 transgenic mice (generated by Ching-Kang Chen, Baylor College of Medicine, Houston TX and made available with permission by Wolfgang Baehr, University of Utah, Salt Lake City, UT) ( 26 ) or CRX-Cre transgenic mice (generated by Tom M. Glaser, University of California, Davis, CA and made available with permission by Anand Swaroop, National Eye Institute, NIH, Bethesda, MD).

Techniques: Labeling, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival

doi: 10.1073/pnas.2010751117

Figure Lengend Snippet: Conditional knockout of STX3 has deleterious effects on photoreceptor terminal organization. TEM of rod and cone terminals are shown from WT (A), Stx3f/f(iCre75) (B), and Stx3f/f(CRX-Cre) (C) retinas. Arrows highlight synaptic ribbons. Arrowheads highlight flat contacts at the base of cone terminals. (D and E) Retinal sections from P21 WT and P15 Stx3f/f(CRX-Cre) retina were labeled for PMCA (red, photoreceptor and bipolar cell synaptic terminals), and either VGLUT1 (green, photoreceptor and bipolar cell synaptic terminals, D) or PKC (green, rod bipolar cells, E), and nuclei are blue (DAPI). Insets show expanded views of boxed areas. Arrows highlight areas of colocalization where rod bipolar dendrites protrude into rod photoreceptor synapses. Arrowheads show photoreceptor terminals receding into the ONL during degeneration. (Scale bar: 400 nm in A–C, 800 nm in B, and 20 µm in D and E.)

Article Snippet: Stx3 flox/flox ( Stx3 f/f ) mice were generated by inGenious Targeting Laboratory, Inc., Ronkonkoma, NY and crossed with iCre75 transgenic mice (generated by Ching-Kang Chen, Baylor College of Medicine, Houston TX and made available with permission by Wolfgang Baehr, University of Utah, Salt Lake City, UT) ( 26 ) or CRX-Cre transgenic mice (generated by Tom M. Glaser, University of California, Davis, CA and made available with permission by Anand Swaroop, National Eye Institute, NIH, Bethesda, MD).

Techniques: Knock-Out, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival

doi: 10.1073/pnas.2010751117

Figure Lengend Snippet: STX3 knockout impairs photoreceptor function. Full-field scotopic (A and C) and photopic (B and D) ERG was performed. (A and B) Representative ERG waveforms. (C and D) The amplitude of maximum scotopic a- and b-waves, and maximum photopic b-waves were plotted as mean ± SEM. n = 3 to 6 animals for each genotype. **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-way ANOVA with Tukey’s post hoc comparison.

Article Snippet: Stx3 flox/flox ( Stx3 f/f ) mice were generated by inGenious Targeting Laboratory, Inc., Ronkonkoma, NY and crossed with iCre75 transgenic mice (generated by Ching-Kang Chen, Baylor College of Medicine, Houston TX and made available with permission by Wolfgang Baehr, University of Utah, Salt Lake City, UT) ( 26 ) or CRX-Cre transgenic mice (generated by Tom M. Glaser, University of California, Davis, CA and made available with permission by Anand Swaroop, National Eye Institute, NIH, Bethesda, MD).

Techniques: Knock-Out, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival

doi: 10.1073/pnas.2010751117

Figure Lengend Snippet: Mislocalized OS proteins colocalize in the absence of STX3. (A and B) P15 retinal sections immunolabeled for PRPH2 (green), ROM1 (red), and rhodopsin (RHO, blue). Nuclei are gray (DAPI) in overlay panels. Arrowheads highlight colocalization of all three proteins; arrows show regions where PRPH2/rhodopsin colocalize in the absence of ROM1. (B) Three-dimensional reconstructions of confocal stacks (YX planes and YZ planes of 1-µm thickness) highlighting the OSs and ISs (Top) or the ONL and OPL (Bottom). Arrows highlight regions where all three proteins colocalize. (C–E) Retinal sections were immunolabeled for PRPH2 (green) and acetylated α-tubulin (red) in C; rhodopsin (red) and acetylated α-tubulin (gray) in D; and PRPH2 (green), ROM1 (red), and acetylated α-tubulin (gray) in E. Nuclei are blue (DAPI). Arrows indicate regions of colocalization. Small panels to the Right of each main panel show expanded views of microtubules. Letters above each panel indicate which channels are shown. R, red; G, green; B, blue; Gy, gray. (Scale bars: 10 µm in A and 20 µm in C–E.) For 3D reconstructions, each grid box is 1 μm.

Article Snippet: Stx3 flox/flox ( Stx3 f/f ) mice were generated by inGenious Targeting Laboratory, Inc., Ronkonkoma, NY and crossed with iCre75 transgenic mice (generated by Ching-Kang Chen, Baylor College of Medicine, Houston TX and made available with permission by Wolfgang Baehr, University of Utah, Salt Lake City, UT) ( 26 ) or CRX-Cre transgenic mice (generated by Tom M. Glaser, University of California, Davis, CA and made available with permission by Anand Swaroop, National Eye Institute, NIH, Bethesda, MD).

Techniques: Immunolabeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival

doi: 10.1073/pnas.2010751117

Figure Lengend Snippet: PRPH2 interacts with SNARE proteins in the absence of STX3. (A) Retinal extracts from WT, Prph2−/−, and Stx3f/f(CRX-Cre) mice underwent immunoprecipitation with PRPH2 antibodies (RDS-CT) and blots were probed for STX3, SNAP25, STXBP1, and PRPH2. I, input; FT, flow through (unbound); E, bound fraction eluted from beads; and B, bound fraction on beads. (B–E) Retinal sections immunolabeled for PRPH2 (green) and SNAP25 (red) in B, PRPH2 (green) and STXBP1 (red) in C, rhodopsin (RHO, green) and SNAP25 (red) in D, and STXBP1 (green) and SNAP25 (red) in E. Nuclei are blue (DAPI). Images shown are projection images from confocal microscope (63×). Arrows indicate regions of colocalization. (F–I) GST fusion constructs were generated, consisting of the PRPH2 C-terminal (F), the STX3 SNARE domain (H), or GST alone as control (G and I). GST proteins were incubated with retinal extracts as indicated at the Top Right of each panel. Each blot has input (total retinal lysate) on the Left and eluents from the GST pulldowns on the Right. Rhodopsin is present in large >250-kDa aggregates because the beads are boiled to elute sample, and input samples are prepared the same way to promote consistency. (Scale bars: 10 μm.)

Article Snippet: Stx3 flox/flox ( Stx3 f/f ) mice were generated by inGenious Targeting Laboratory, Inc., Ronkonkoma, NY and crossed with iCre75 transgenic mice (generated by Ching-Kang Chen, Baylor College of Medicine, Houston TX and made available with permission by Wolfgang Baehr, University of Utah, Salt Lake City, UT) ( 26 ) or CRX-Cre transgenic mice (generated by Tom M. Glaser, University of California, Davis, CA and made available with permission by Anand Swaroop, National Eye Institute, NIH, Bethesda, MD).

Techniques: Immunoprecipitation, Immunolabeling, Microscopy, Construct, Generated, Control, Incubation

Journal: bioRxiv

Article Title: Mono-ubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

doi: 10.1101/164996

Figure Lengend Snippet: (A) Endogenous Stx3 was immunoprecipitated (IP) from Caco2 cells and immunoblotted (IB) with an antibody to ubiquitin (Ub) and Stx3. Nonspecific rabbit IgG antibody was used for control (lane C). (B) HEK293T cells were transfected with untagged Stx3 and myc - ubiquitin constructs. Lysates were subjected to IP with an anti - Stx3 antibody and analyzed by IB with anti - myc antibody. Cell lysates used as input for the IP were blotted with anti - Stx3 antibody. The ubiquitinated Stx3 band identified by both anti - myc and anti - Stx3 antibodies is indicated by arrow. (C) Doxycycline inducible MDCK cells expressing Stx3 or Stx4, both myc - tagged, were lysed, immunoprecipitated with anti - myc antibodies, and analyzed by immunoblot with antibodies to myc or Ubi. Bottom panel: total lysates used for the IP were blotted with anti - myc antibody. (D) Untransfected MDCK cells or MDCK cells stably expressing double myc - tagged Stx3 were treated with 10μM ALLN (+ ALLN) or without (- ALLN) for 16h. Lysates were subjected to IP with anti - myc antibody and analyzed by IB with anti - myc antibody.

Article Snippet: QuickChange Site - directed mutagenesis kit (Agilent Technologies) was used to generate mutations in the pcDNA4 - Stx3 expression construct per manufacturers instructions. pGFP - C2 - hRab5A and pGFP - C2 - hRab5A - Q79L were gifts from Dzwokai Ma (University of California, Santa Barbara). pEGFP - GPRC5B plasmid was a gift from Keith Mostov and Sang - Ho Kwon (University of California, San Francisco). mCherry - Rab5CA(Q79L) was a gift from Sergio Grinstein (Addgene plasmid # 35138) ( ). shRNA - resistant c - myc - Stx3 (GenScript, New Jersey, USA) and c - myc - Stx3 - 5R constructs were extended with Gateway ® recombination sequences and transferred via pDONR201 (Invitrogen) to the lentiviral plasmid pLenti - PGK - Neo - DEST(w531 - 1) ( ) (obtained through Addgene, Massachusetts, USA).

Techniques: Immunoprecipitation, Ubiquitin Proteomics, Control, Transfection, Construct, Expressing, Western Blot, Stable Transfection

Journal: bioRxiv

Article Title: Mono-ubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

doi: 10.1101/164996

Figure Lengend Snippet: (A) Schematic representation of Stx3 wild - type and mutant constructs used for expression. Residues in the polybasic juxtamembrane region (PB) of wild - type Stx3 are shown. Two myc epitope tags (white circles) and one His6 tag (black circle) were added to the COOH termini; PB, polybasic; TM, transmembrane domain. (B) HEK293T cells were transiently transfected with the indicated constructs, plated for 24h and incubated with or without ALLN. Lysates were subjected to IP with anti - myc antibody and analyzed by IB with anti - Ubi. Bottom: Total lysates used for IP blotted with anti - myc antibody. Cells without construct transfection (lane C) serve as a negative control for the IP. (C) Sequence alignment of the juxtamembrane region of human plasma membrane syntaxins 1A, 2, 3 and 4, SwissProt accession numbers Q16623 , P32856 , Q13277 , Q12846 respectively. (D) Stx3 lysine mutant constructs used in this study (region 241 - 268 is shown). Lysine residues mutated to arginine are in bold letters. (E) Anti - myc Western Blot of HEK293T treated with and without ALLN transiently expressing lysine mutants subjected to anti - myc IP. (F) Two different stable clones of DOX inducible MDCK cells stably transfected for Stx3 - 5R, c14 and c21, were subjected to IP with anti - myc antibody and IB with antibodies against Ubi, SNAP - 23, and munc18b. (G) Immunocytochemistry of DOX - induced Stx3, Stx3 - 5R, or Stx3 - 6R (green) expresses in polarized MDCK cells cultured on Transwell filters. Scale bar: 10μm.

Article Snippet: QuickChange Site - directed mutagenesis kit (Agilent Technologies) was used to generate mutations in the pcDNA4 - Stx3 expression construct per manufacturers instructions. pGFP - C2 - hRab5A and pGFP - C2 - hRab5A - Q79L were gifts from Dzwokai Ma (University of California, Santa Barbara). pEGFP - GPRC5B plasmid was a gift from Keith Mostov and Sang - Ho Kwon (University of California, San Francisco). mCherry - Rab5CA(Q79L) was a gift from Sergio Grinstein (Addgene plasmid # 35138) ( ). shRNA - resistant c - myc - Stx3 (GenScript, New Jersey, USA) and c - myc - Stx3 - 5R constructs were extended with Gateway ® recombination sequences and transferred via pDONR201 (Invitrogen) to the lentiviral plasmid pLenti - PGK - Neo - DEST(w531 - 1) ( ) (obtained through Addgene, Massachusetts, USA).

Techniques: Mutagenesis, Construct, Expressing, Transfection, Incubation, Negative Control, Sequencing, Clinical Proteomics, Membrane, Western Blot, Clone Assay, Stable Transfection, Immunocytochemistry, Cell Culture

Journal: bioRxiv

Article Title: Mono-ubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

doi: 10.1101/164996

Figure Lengend Snippet: (A) Immunofluorescence microscopy showing reduced colocalization of Stx3 - 5R with LAMP - 2 compared to wild - type Stx3. Cells were cultured on coverslips, treated with DOX for 16h and stained with Stx3 (green) and LAMP - 2 (red) antibodies. Intensity of pixels along white lines in micrographs for each channel plotted in graphs under micrographs. Scale bar: 10μm. (B) Wild - type Stx3 or Stx3 - 5R (c14) expression was induced with DOX for 16h in stably transfected MDCK cells grown at confluence for 4 days on Transwell filters. For endocytosis assay anti - myc 9E10 ascites was added to apical or basolateral media and incubated at 4°C before being washed off and incubated at 37°C for indicated times. Cells were fixed and stained for Stx3 (myc), green; M6PR, red; and nuclei (DAPI), blue. Scale bar: 10 μm. (C) MDCK cells stably expressing Stx3 or Stx3 - 5R were grown on coverslips and transiently transfected with GFP - Rab5 or GFP - Rab5Q79L. Twenty - four hours after transfection, Stx3 or Stx3 - 5R expression was induced with DOX for 16h. Cells were fixed and processed for immunocytochemistry. Stx3 (myc), red; Rab5, green; and nuclei (DAPI), blue. Scale bar: 10μm. (D) MDCK cells were grown on coverslips and transiently transfected with mCherry - Rab5 - Q79L, GFP - GPRC5B, and either Stx3 or Stx3 - 5R. Thirty - six hours after transfection cells were fixed and processed for immunocytochemistry. Stx3 (myc), blue; Rab5, red; and GPRC5B, green. Scale bar: 10μm.

Article Snippet: QuickChange Site - directed mutagenesis kit (Agilent Technologies) was used to generate mutations in the pcDNA4 - Stx3 expression construct per manufacturers instructions. pGFP - C2 - hRab5A and pGFP - C2 - hRab5A - Q79L were gifts from Dzwokai Ma (University of California, Santa Barbara). pEGFP - GPRC5B plasmid was a gift from Keith Mostov and Sang - Ho Kwon (University of California, San Francisco). mCherry - Rab5CA(Q79L) was a gift from Sergio Grinstein (Addgene plasmid # 35138) ( ). shRNA - resistant c - myc - Stx3 (GenScript, New Jersey, USA) and c - myc - Stx3 - 5R constructs were extended with Gateway ® recombination sequences and transferred via pDONR201 (Invitrogen) to the lentiviral plasmid pLenti - PGK - Neo - DEST(w531 - 1) ( ) (obtained through Addgene, Massachusetts, USA).

Techniques: Immunofluorescence, Microscopy, Cell Culture, Staining, Expressing, Stable Transfection, Transfection, Endocytosis Assay, Incubation, Immunocytochemistry

Journal: bioRxiv

Article Title: Mono-ubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

doi: 10.1101/164996

Figure Lengend Snippet: (A) Exosomes from 60mL of human urine (from two volunteers 1 and 2) were isolated by differential ultracentrifugation. Indicated volume amount from a total volume of 50μL were analyzed by immunoblotting for TSG101 (exosome positive marker), Na/K ATPase (exosome negative marker) ( ; ), Stx3, and munc18b. Total lysates from Caco - 2 cells were used as antibody control (L) (B) Human urine exosomes were analyzed for the indicated plasma membrane syntaxins. Lysates from MDCK cells with stable expression for the respective syntaxins were used as positive controls. (C) Exosomes from Caco - 2 conditioned media were isolated and analyzed as above. (D) HEK293T cells were transiently transfected with Stx3, Stx3 - 5R, and Stx4 (all myc tagged) plasmids. Lane C represents cells without transfection. Forty - eight hours after transfection exosomes from the media and cell lysates were analyzed by immunoblotting with anti - myc, anti - calnexin (exosome negative marker) and anti - flotillin - 1 (exosome positive marker). As a specificity control, Stx4 was included which was not appreciably secreted in exosomes. (E) DOX induced MDCK expressing Stx3 or Stx3 - 5R were transiently transfected with GFP - GPRC5B. Exosomes were purified from media after 48h and subjected to immunoblotting. Performed in triplicate. (F) Quantification of relative intensity of GPRC5B bands in Stx3 or Stx3 - 5R exosome samples from (E). Error bars represent SEM of experiment performed in triplicate. *P< 0.05, Student’s unpaired t - test. (G) Immunoblot of MeWo cells transduced with lentivirus delivering shRNA #304, targeting Stx3, or shRNA scrambled control. (H) Table showing concentration of exovesicles in media collected from cells in panel (G).

Article Snippet: QuickChange Site - directed mutagenesis kit (Agilent Technologies) was used to generate mutations in the pcDNA4 - Stx3 expression construct per manufacturers instructions. pGFP - C2 - hRab5A and pGFP - C2 - hRab5A - Q79L were gifts from Dzwokai Ma (University of California, Santa Barbara). pEGFP - GPRC5B plasmid was a gift from Keith Mostov and Sang - Ho Kwon (University of California, San Francisco). mCherry - Rab5CA(Q79L) was a gift from Sergio Grinstein (Addgene plasmid # 35138) ( ). shRNA - resistant c - myc - Stx3 (GenScript, New Jersey, USA) and c - myc - Stx3 - 5R constructs were extended with Gateway ® recombination sequences and transferred via pDONR201 (Invitrogen) to the lentiviral plasmid pLenti - PGK - Neo - DEST(w531 - 1) ( ) (obtained through Addgene, Massachusetts, USA).

Techniques: Isolation, Western Blot, Marker, Control, Clinical Proteomics, Membrane, Expressing, Transfection, Purification, Transduction, shRNA, Concentration Assay

Journal: bioRxiv

Article Title: Mono-ubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

doi: 10.1101/164996

Figure Lengend Snippet: BJ1 cells expressing shRNA - resistant - STX3 or shRNA - resistant - Stx3 - 5R constructs were transduced with scrambled control and STX3 #304 shRNAs and infected with RecCMV at 0.3 moi. (A) Immunoblot analysis of these cells at 4 days post infection with antibodies against STX3 and actin. (B) Flow cytometry analysis for GFP expression. (C) At 5 dpi, supernatants were harvested and the number of extracellular infectious viruses was determined on fresh BJ1 cells. Data are means plus standard deviations of experiment performed in quadruplicate.

Article Snippet: QuickChange Site - directed mutagenesis kit (Agilent Technologies) was used to generate mutations in the pcDNA4 - Stx3 expression construct per manufacturers instructions. pGFP - C2 - hRab5A and pGFP - C2 - hRab5A - Q79L were gifts from Dzwokai Ma (University of California, Santa Barbara). pEGFP - GPRC5B plasmid was a gift from Keith Mostov and Sang - Ho Kwon (University of California, San Francisco). mCherry - Rab5CA(Q79L) was a gift from Sergio Grinstein (Addgene plasmid # 35138) ( ). shRNA - resistant c - myc - Stx3 (GenScript, New Jersey, USA) and c - myc - Stx3 - 5R constructs were extended with Gateway ® recombination sequences and transferred via pDONR201 (Invitrogen) to the lentiviral plasmid pLenti - PGK - Neo - DEST(w531 - 1) ( ) (obtained through Addgene, Massachusetts, USA).

Techniques: Expressing, shRNA, Construct, Transduction, Control, Infection, Western Blot, Flow Cytometry