Journal: Molecular and Cellular Biology

Article Title: Stxbp4 Regulates ΔNp63 Stability by Suppression of RACK1-Dependent Degradation

doi: 10.1128/mcb.00449-09

Figure Lengend Snippet: FIG. 2. The physical interaction between p63 and Stxbp4. (A) Exogenously expressed p63 and Stxbp4 interact with each other. H1299 cells were transfected with plasmids encoding Flag-tagged Stxbp4 and Myc-tagged Np63 or Np63. Cell lysates were immunoprecipitated (IP) with anti-Myc antibody and immunoblotted with anti-Myc and anti-Flag antibodies, respectively. A total of 15% of the lysate was used in the input sample. (B) The WW domain of Stxbp4 and the PPPPY motif of Np63 are required for Stxbp4 interaction with p63. Shown on top is a schematic illustration of the modular structure of Stxbp4 and Np63 and corresponding deletion or point mutation constructs. H1299 cells were transfected with the indicated plasmids and processed for immunoblotting as shown in panel A. A total of 10% of the lysate was used in the input sample. (C) Endogenous p63 proteins interact with exogenous Stxbp4. A Flag-tagged Stxbp4 construct was transfected into Scaber and HaCaT cells and after 24 h, cell lysates were prepared and immunoprecipitated with anti-Np63 antibody or control rabbit IgG, followed by immunoblotting with anti-Flag and anti-p63 (4A4) antibodies, respectively. A total of 2% of the lysate was used in the input sample. (D) Endogenous Stxbp4 and p63 interact. Lysates of HaCaT cells were immunoprecipitated with anti-Np63, anti-p63, or control rabbit IgG, followed by immunoblotting with anti-p63 (4A4) and anti-Stxbp4 antibodies, respectively. A total of 1% of the lysate was used as the input sample. (E) Stxbp4 can directly bind p63 in vitro as indicated by far-Western analysis. His-tagged p63 proteins were purified from baculovirus-infected sf9 cells, subjected to SDS-PAGE, and then visualized by silver staining (right panel). GST or GST-Stxbp4 proteins were expressed in DH5 cells by IPTG (isopropyl--D-thiogalactopyranoside) induction, then separated by SDS-PAGE, and stained by Coomassie blue (left panel). The same lysate was resolved on two parallel gels, which were transferred to a nitrocellulose membrane, denatured by 6 M guanidinium HCl, and renatured by serial dilutions of guanidinium HCl as described in Materials and Methods. The membrane was then incubated with or without 0.5 mg/ml purified His-tagged p63 (the middle two panels) and immunoblotted with anti-His antibody. Molecular weights (in thousands) are indicated on the left.

Article Snippet: The Stxbp4 polyclonal antibody was also raised commercially (Proteintech Group) against purified GST-Stxbp4 (1–207).

Techniques: Transfection, Immunoprecipitation, Mutagenesis, Construct, Western Blot, Control, In Vitro, Infection, SDS Page, Silver Staining, Staining, Membrane, Incubation

Journal: Molecular and Cellular Biology

Article Title: Stxbp4 Regulates ΔNp63 Stability by Suppression of RACK1-Dependent Degradation

doi: 10.1128/mcb.00449-09

Figure Lengend Snippet: FIG. 3. Stxbp4, like Np63, is essential for keratinocyte prolifera- tion. HaCaT human keratinocytes were transfected with control lucif- erase (Luc), two Stxbp4, or two p63 siRNAs, as indicated. Cells were collected 72 h later and processed for immunoblotting (A) or FACS analysis (B). The population of cells in the S phase is shown as an index of cell proliferation. Molecular weights (in thousands) are indicated on the left in panel A.

Article Snippet: The Stxbp4 polyclonal antibody was also raised commercially (Proteintech Group) against purified GST-Stxbp4 (1–207).

Techniques: Transfection, Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Stxbp4 Regulates ΔNp63 Stability by Suppression of RACK1-Dependent Degradation

doi: 10.1128/mcb.00449-09

Figure Lengend Snippet: FIG. 4. Stxbp4 regulates Np63 protein stability. (A) HaCaT cells were transfected with siRNAs as described in the legend for Fig. 3. At 72 h, cells were collected and processed for immunoblotting (W.B.) to detect p63, Stxbp4, p53, and actin (top four panels). The RNA transcript levels of p63, stxbp4, and actin were analyzed by RT-PCR (bottom three panels). (B) The dose-dependent p63 destabilization by Stxbp4 siRNA is shown, using Stxbp4-2 siRNA as an example. (C) At 62 h after siRNA transfection, HaCaT cells were treated with 20 g/ml cycloheximide (CHC) and collected at the indicated time points to detect p63, Stxbp4, and actin by Western blotting. The results after densitometric analysis and normalization based on actin levels were graphed (right). (D) At 42 h after siRNA transfection, HaCaT cells were treated with or without MG132 for 8 h and were subjected to immunoblotting. (E) Scaber cells were transfected with control luciferase (Luc) or two Stxbp4 siRNAs for 72 h and analyzed by immunoblotting. (F) U2OS cells were transfected with Flag-tagged Np63 alone or together with an increasing amount of Flag-tagged Stxbp4. Cells were collected 44 h later and were processed for immunoblotting using the indicated antibodies. Molecular weights (in thousands) are indicated on the left of panels B, C, D, E, and F.

Article Snippet: The Stxbp4 polyclonal antibody was also raised commercially (Proteintech Group) against purified GST-Stxbp4 (1–207).

Techniques: Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Luciferase

Journal: Molecular and Cellular Biology

Article Title: Stxbp4 Regulates ΔNp63 Stability by Suppression of RACK1-Dependent Degradation

doi: 10.1128/mcb.00449-09

Figure Lengend Snippet: FIG. 5. Itch-mediated Np63 degradation is inhibited by Stxbp4, but endogenous Itch is unlikely to be involved in Np63 degradation. (A) 293 cells were transfected with Flag-tagged Np63 (0.1 g) and Myc-tagged Itch at the indicated ratios (0.1 g, 0.3 g, 1 g, and 1.5 g). (B) 293 cells were transfected with Flag-tagged Np63 (0.1 g) and Myc-tagged Itch (0.3 g), together with different amounts of Flag-tagged Stxbp4 plasmid (0.3 g, 1 g or 3 g). At 48 h after transfection, cells shown in panels A and B were processed for immunoblotting to detect p63, Itch, Stxbp4, and actin, as indicated. (C) HaCaT cells were transfected with control (luciferase [Luc]), Stxbp4 (Stxbp4-1), or Itch (Itch-1 and Itch-2) siRNAs. Cells were collected 72 h later and processed for immunoblotting. Molecular weights (in thousands) are indicated on the left.

Article Snippet: The Stxbp4 polyclonal antibody was also raised commercially (Proteintech Group) against purified GST-Stxbp4 (1–207).

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Luciferase

Journal: Molecular and Cellular Biology

Article Title: Stxbp4 Regulates ΔNp63 Stability by Suppression of RACK1-Dependent Degradation

doi: 10.1128/mcb.00449-09

Figure Lengend Snippet: FIG. 6. Np63 destabilization in the absence of Stxbp4 is dependent on RACK1 pathway. (A) U2OS cells were transfected with 0.1 g plasmid expressing Flag-tagged Np63 alone and with increasing amounts of plasmid expressing T7-tagged RACK1 (0.3 g, 1 g, or 3 g). Cells were collected 24 h later and processed for immunoblotting. (B) U2OS cells were transfected with Flag-tagged Np63 (0.1 g) and T7-tagged RACK1 (2 g), together with increasing amounts of Flag-tagged Stxbp4 (0.5 g, 1 g, or 2 g). At 40 h after transfection, cells were processed for immunoblotting using the indicated antibodies. (C) HaCaT cells were transfected with control (luciferase [Luc]), Stxbp4 (Stxbp4-1), RACK1 (Rack1-KD1) siRNAs, or siRNAs against both Stxbp4 and RACK1. Cells were processed for immunoblotting 72 h after transfection. Molecular weights (in thousands) are indicated on the left.

Article Snippet: The Stxbp4 polyclonal antibody was also raised commercially (Proteintech Group) against purified GST-Stxbp4 (1–207).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Luciferase

Journal: Molecular and Cellular Biology

Article Title: Stxbp4 Regulates ΔNp63 Stability by Suppression of RACK1-Dependent Degradation

doi: 10.1128/mcb.00449-09

Figure Lengend Snippet: FIG. 7. Np63 and Stxbp4 are downregulated upon DNA damage. (A) HaCaT cells were treated with 300 nM camptothecin (CPT) for the indicated time points. Cells were collected for both immunoblotting (W.B.) (top two panels) and RT-PCR with primers specific for Np63 or -actin mRNAs (bottom two panels). (B) An H1299 Tet-off cell line was cultured in medium without tetracycline to induce Np63 expression for 24 h. Cells were then treated with 300 nM CPT and 30 etoposide (ETP) for 21 h and processed for immunoblotting. (C and D) HaCaT cells were transfected with siRNAs for RACK1, Itch, or control (luciferase [Luc]). At 48 h after transfection, cells were treated with either 30 ETP or 300 nM CPT for 24 h and processed for immunoblotting using the indicated antibodies. DMSO, dimethyl sulfoxide. HaCaT cells were treated with either 30 ETP or 300 nM CPT for 24 h (E) or with 300 nM CPT for the indicated time points (F). Cells were processed for immunoblotting to detect Stxbp4, p63, and actin. Molecular weights (in thousands) are indicated on the left.

Article Snippet: The Stxbp4 polyclonal antibody was also raised commercially (Proteintech Group) against purified GST-Stxbp4 (1–207).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Expressing, Transfection, Control, Luciferase

Journal: Molecular and Cellular Biology

Article Title: Stxbp4 Regulates ΔNp63 Stability by Suppression of RACK1-Dependent Degradation

doi: 10.1128/mcb.00449-09

Figure Lengend Snippet: FIG. 8. Models for the regulation of Np63 stability under normal growth conditions and in response to DNA damage. (A) In resting conditions, Np63 is expressed at a relatively high level to promote cell proliferation and/or survival. Its basal level is maintained by Stxbp4, which suppresses RACK1-mediated degradation. (B) Following DNA damage, Stxbp4 itself is downregulated, allowing RACK1 to target Np63 for degradation, which eventually leads to cell cycle arrest or cell death.

Article Snippet: The Stxbp4 polyclonal antibody was also raised commercially (Proteintech Group) against purified GST-Stxbp4 (1–207).

Techniques:

Journal: Developmental Cell

Article Title: ORF3a of SARS-CoV-2 promotes lysosomal exocytosis-mediated viral egress

doi: 10.1016/j.devcel.2021.10.006

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-STX4 (clone E6W7B) , Cell Signaling Technology , Cat# 67657S; N/A.

Techniques: Virus, Recombinant, Protease Inhibitor, cDNA Synthesis, Cell Fractionation, Sequencing, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

doi: 10.1038/s41467-024-46953-x

Figure Lengend Snippet: A SNAP29, SNAP23, STX3, SEC22B, and FKBP5 co-immunoprecipitation (SKA2 IP) and whole cell extract (WCE) in hippocampus (HIP), prefrontal cortex (PFC) and amygdala (AMY) samples of mice ( n = 8). B HIS pull down assay (replicated in 3 independent in vitro experiments). DDK(Flag)-tagged SNAP23, SNAP29, Syntaxin3 or Syntaxin4 was incubated with purified magnetic beads-HIS-tagged SKA2 or magnetic beads-HIS protein alone. After incubation, bead bound proteins were eluted at room temperature (RT) or at 95 °C and subjected to western blot analysis using antibodies against HIS and FLAG. Input lane contains HIS alone (left) or HIS-tagged SKA2 (right). C – M SIM-A9 cells transfected with SKA2, FKBP5 or their respective controls, were harvested 24 h later. After immunoprecipitation (IP) of protein complexes, input and co-IP proteins were quantified by western blotting. C , F , I , K Representative blots of ( D , E , G , H , J , L , M ). Graphs display quantification of SNAP29/SEC22B, STX3/SEC22B, SKA2/SNAP29, FKBP5/SEC22B protein association after SEC22B or SNAP29 IP (unpaired two tailed t-test: ( D ) t 6 = 8.945, p < 0.0001, ( E ) t 6 = 12.94, p < 0.0001, ( G ) t 6 = 6.056, p = 0.0009, ( H ) t 6 = 5.554, p = 0.0014; one-way ANOVA: ( J ) F 2, 9 = 17.28, p = 0.0008, Tukey’s post hoc test: ctrl vs. FKBP5-OE, p = 0.0743, ctrl vs. FKBP5-KO, p = 0.0218, FKBP5-OE vs. FKBP5-KO, p = 0.0006; unpaired two tailed t-test: ( L ) t 6 = 10.27, p < 0.0001, ( M ) t 6 = 8.140, p = 0.0002; n = mean derived from four independent in vitro experiments). * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Data are presented as mean + SEM. Source data are provided as a file.

Article Snippet: Purified Syntaxin3-DDK (Origene, TP300658), SNAP29-DDK (Origene, TP302179), Syntaxin4 (Origene, TP300347), SNAP23-DDK (Origene, TP301596) or correspondingly SEC22B-HIS (Origene, AR50533PU-S) (100 ng) was used for the binding reaction.

Techniques: Immunoprecipitation, Pull Down Assay, In Vitro, Incubation, Purification, Magnetic Beads, Western Blot, Transfection, Co-Immunoprecipitation Assay, Two Tailed Test, Derivative Assay

Journal: Molecular Biology of the Cell

Article Title: Munc13-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses

doi: 10.1091/mbc.E15-05-0283

Figure Lengend Snippet: Munc13-4 binds to syntaxin 7 and VAMP8 in a calcium-dependent manner. (A) Binding assays for Munc13-4 and SNARE proteins. The binding of Flag-Munc13-4 to several GFP-tagged SNARE proteins or GFP (E. Vec) was analyzed by the TR-FRET assay as described in Materials and Methods . The reactions were carried out using 293T cell lysates. Where indicated, the reactions were performed in the presence of 100 μM CaCl 2 or 200 μM EGTA. Triplicates of one experiment, representative of at least four experiments. * p < 0.05; NS, not significant. STX, syntaxin. (B) Binding assays of Munc13-4 and syntaxin 7 in the presence of 100 μM EGTA or CaCl 2 at the indicated total calcium concentration. Emission ratios were normalized to sample NA (no addition). The results are expressed as mean ± SEM. NS, not significant. *** p < 0.001 and * p < 0.05 vs. EGTA. (C, D) Coimmunoprecipitation assays were carried out using anti-Flag beads and 293T cell lysates expressing the indicated proteins. The cells were disrupted by nitrogen cavitation in either relaxation buffer (C) or RIPA buffer (D) as described in Materials and Methods . Western blots are representative of at least three experiments with similar results. The asterisk indicates truncations. (E, F) Analysis of the binding of Munc13-4 WT or mutants C2A*, C2B*, and C2A*C2B* to syntaxin 7 (E) or VAMP8 (F). C2A* includes point mutations D127 and D133 to alanines in the Munc13-4 C2A domain, which knocks out the Ca 2+ -binding sites in this domain; C2B* includes point mutations D941 and D947 to alanines to knock out the Ca 2+ -binding sites in the C2B domain. C2A*C2B* includes four D → A mutations corresponding to both C2 domains. NS, not significant. ** p < 0.001. (G) Coimmunoprecipitation assays were performed as in C, except that reactions were performed in the presence or absence of 100 μM calcium (Ca 2+ ), and, where indicated, Munc13-4 C2 mutants were used instead of WT Munc13-4.

Article Snippet: GFP-Vamp8, syntaxin 7, syntaxin 17, syntaxin 18, and Vti1b were obtained from Addgene (Cambridge, MA).

Techniques: Binding Assay, Concentration Assay, Expressing, Western Blot, Knock-Out

Journal: Molecular Biology of the Cell

Article Title: Munc13-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses

doi: 10.1091/mbc.E15-05-0283

Figure Lengend Snippet: Syntaxin 7 and Munc13-4 colocalize in LAMP1-positive granules in neutrophils, but the subcellular localization of syntaxin 7 and VAMP8 is independent of Munc13-4. (A) Immunofluorescence analysis of endogenous syntaxin 7 (STX7; green), the late endosomal marker LAMP1 (red), and Munc13-4 (violet) in wild-type neutrophils. Arrowheads indicate examples of vesicles that are positive for all three stainings. Cell nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). (B) Immunofluorescence analysis of endogenous syntaxin 7 (green), the lysosome-related organelle marker MPO (red), and Munc13-4 (violet) in wild-type neutrophils. (C) Colocalization analysis of LAMP1 with either syntaxin 7 or Munc13-4 using 29 and 38 cells, respectively. (D, E) High-resolution STORM analysis of the localization of endogenous Munc13-4 and syntaxin 7 (D) or LAMP1 (E) in wild-type cells. Whereas syntaxin 7 and Munc13-4 are detected adjacent to each other (arrowheads, 10–50 nm; D), LAMP1 and Munc13-4 are in close proximity but not always adjacent (arrowheads, estimated distance >50 nm; E). Scale bar, 1 μm. (F) TIRFM analysis of colocalization of syntaxin 7 and Munc13-4 in RPE cells. Scale bar, 10 μm. (G, H) Immunofluorescence analyses of endogenous syntaxin 7 (green) and VAMP8 (violet) with LAMP1 (red; G) or MPO (red; H) in wild-type and Munc13-4–KO neutrophils. Arrowheads indicate examples of vesicles that are positive for all three stainings. Scale bar, 1 μm. (I) Quantification of the colocalization shown in G and H. Calculation of the colocalization coefficient was performed by analyzing at least 38 cells in each group. Results are mean ± SEM. *** p < 0.001.

Article Snippet: GFP-Vamp8, syntaxin 7, syntaxin 17, syntaxin 18, and Vti1b were obtained from Addgene (Cambridge, MA).

Techniques: Immunofluorescence, Marker, Staining

Journal: Molecular Biology of the Cell

Article Title: Munc13-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses

doi: 10.1091/mbc.E15-05-0283

Figure Lengend Snippet: Munc13-4 wild type but not the syntaxin 7 binding–deficient mutant Munc13-4 C2A*C2B* rescues the late endosome enlargement phenotype in Munc13-4–KO cells. (A) WT and Munc13-4–KO ( Jinx ) neutrophils were cotransfected with EGFP-LAMP1 and mCherry-Munc13-4 wild type (Munc13-4 WT), mCherry-Munc13-4 C2A*C2B* mutant (Munc13-4C2A*C2B*), or mCherry empty vector (E. Vec). Quantification of EGFP-LAMP1–positive late endosome diameters was performed as in . Results are mean ± SEMs, with 40, 47, 32, and 42 cells quantified for groups WT with mCherry empty vector, Jinx with mCherry empty vector, Jinx with mCherry-Munc13-4-WT, and Jinx with mCherry-Munc13-4-C2A*C2B* mutant, respectively. *** p < 0.0001. (B) Representative TIRFM images of cells from A. Cells were imaged 6 h after nucleofection. Scale bar, 1 μm.

Article Snippet: GFP-Vamp8, syntaxin 7, syntaxin 17, syntaxin 18, and Vti1b were obtained from Addgene (Cambridge, MA).

Techniques: Binding Assay, Mutagenesis, Plasmid Preparation