Journal: Gastroenterology

Article Title: Pancreatitis-Induced Depletion of Syntaxin 2 Promotes Autophagy and Increases Basolateral Exocytosis.

doi: 10.1053/j.gastro.2018.01.025

Figure Lengend Snippet: Figure 4. STX2 deletion promotes STX3- and STX4–mediated SNARE complex assembly. Dispersed pancreatic acini from WT or STX2-KO mice were stimulated for 30 minutes with indicated doses of CCK-8, and then subjected to immunoprecipitation with antibodies against (A) STX3 or (B) STX4. Representative blots are shown in the left panels. Band intensity ratios of co- immunoprecipitated Munc18b/c, SNAP23, and VAMP8/2 relative to immunoprecipitated STX3 (A) or STX4 (B) are shown in corresponding right panels, expressed as ± SEM of 3 independent experiments. Five percent “input” controls (50 mg total acini lysates) with densitometric analysis shown in Supplementary Figure 4A–D. (C) STX2 blocks STX3 and STX4 binding to cognate VAMP8 and VAMP2. STX2 is co-expressed with STX3 or STX4 in HEK cells; controls were expression of single STX and GST. HEK lysates were then subjected to pull-down with GST-VAMP8 or GST-VAMP2. Band intensity ratios of precipitated STX2, STX3 and STX4, and co-precipitated STX2/STX3, and STX2/ STX4 relative to the 5% inputs are shown in corresponding right panels, expressed as mean ± SEM of 3 independent experiments. *P < .05; **P < .01.

Article Snippet: Samples were then blocked 1 hour in 10% goat serum and immunostained with the primary antibody to STX2 (#ab12369), LAMP2 (#ab13524), Abcam (Cambridge, UK; 1:100), STX4 (1:100), LC3B (sc-16755, Santa Cruz Biotechnology, Santa Cruz, CA, 1:200), anti-TAP antibody (ABIN1173333, Antibodies Online, Atlanta, GA; 1:100), anti-clathrin (1:100) or anti-Atg16L1 (1:100).

Techniques: CCK-8 Assay, Immunoprecipitation, Binding Assay, Expressing

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 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: