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: Nature Communications

Article Title: Stress-primed secretory autophagy promotes extracellular BDNF maturation by enhancing MMP9 secretion

doi: 10.1038/s41467-021-24810-5

Figure Lengend Snippet: a Results of automated literature mining of FKBP51 interactors in association to “autophagy”, “proteostasis” and “ubiquitin-proteasome system” (UPS) or none of them. b Western blotting of FKBP51 and SEC22B in GFP-tagged SEC22B co-IP (GFP-IP) and whole-cell extract (WCE) as control; c Western blotting of FKBP51 and SEC22B in FLAG-tagged FKBP51 co-IP (FLAG-IP) and WCE as control. d Western blotting of FKBP51, TRIM16, LC3B, and CTSD in FLAG-tagged FKBP51 co-IP (FLAG-IP) and WCE as control. e Western blotting for TRIM16, CTSD, and SEC22B in FLAG-tagged TRIM16 co-IP (FLAG-IP) and WCE as control performed in WT and FKBP5 KO SH-SY5Y cells. f Quantifications of e with n = 3 biologically independent samples. g Western blotting of TRIM16, CTSD, and SEC22B in FLAG-tagged TRIM16 co-IP (FLAG-IP) and WCE as control performed in cells treated with 100 nM dexamethasone or vehicle for 4 h. h Quantifications of g with n = 3 biologically independent samples. i Western blotting for FKBP51, SNAP23, SNAP29, STX3, and STX4 in FLAG-tagged FKBP51 co-IP (FLAG-IP) and WCE as control. j Western blotting of SEC22B, SNAP23, SNAP29, STX3, and STX4 in GFP-tagged SEC22B co-IP (GFP-IP) and WCE as control performed in WT and FKBP5 KO cells treated with 100 nM dexamethasone or vehicle for 4 h. k Quantifications of j with n = 3 biologically independent samples. b – k All experiments were performed in SH-SY5Y cells. l Schematic model of the interactions of FKBP51 in the secretory autophagy pathway. Unpaired, one-tailed t -tests were performed for all quantifications; ns not significant, ** P < 0.01, *** P < 0.001. Data shown as mean ± s.e.m. Ab antibody, Fc fold change.

Article Snippet: FLAG (1:7000, Rockland, 600-401-383), FKBP51 (1:1000, Bethyl, A301-430A), FKBP5 (D5G2) (1:1000, Cell Signaling, #12210), Actin (1:5000, Santa Cruz Biotechnology, sc-1616), GAPDH (1:8000, Millipore CB1001), TRIM16 (1:1000, Bethyl A301-160A), CTSD (for Human) (1:50, Abcam, ab6313), CTSD (for Mouse) (1:50, Abcam, ab207549), SNAP29 (1:1000, Sigma, SAB1408650), SNAP23 (1:1000, Sigma, SAB2102251), STX3 (1:1000, Sigma, SAB2701366), STX4 (1:1000, Cell Signaling, #67657), GAL8 (1:1000, Santa Cruz, sc-28254), GAL3 (1:1000, Santa Cruz, sc-32790), SEC22B (1:1000, Abcam, ab181076 and 1:1000, Thermo Fisher, OSS00040W), Vinculin (1:1000, Cell Signaling, #13901)

Techniques: Ubiquitin Proteomics, Western Blot, Co-Immunoprecipitation Assay, Control, One-tailed Test

Journal: bioRxiv

Article Title: Dual Ligand Cooperation at the Plasma Membrane Drives Transport of Engineered Small Extracellular Vesicles Across Brain Endothelial Cells

doi: 10.64898/2026.01.21.700773

Figure Lengend Snippet: sEVs-HA-Tf accumulate at the basolateral membrane of BECs. (a) Scheme illustrating the trancystosis steps of sEVs or sEVs-HA-Tf in BECs. (b.1) Expression of syntaxin-4 in human BECs along the z-axis. xy-slice at the level of the basolateral (z-stack:2/32) and apical (z-stack:17/32) membranes. Scale bar: 40 µm. (b.2) Number of Syntaxin-4 foci along the z-axis. (c.1) Representative image of Transferrin-594 conjugate, acquired at the bottom most plane of the glass slide, on STED mode using a single point scanning confocal Stellaris 8 (Leica) microscope. (c.2) Representative images of EVs, EVs-HA and EVs-HA-Tf, acquired with similar settings. Scale bar: 1 µm. (c.3) Measured area (μm2) of the encircled organelle structures depicted in c.1 and c.2. Results are expressed as mean±SEM (3 fields analyzed per experimental condition across 105-135 cells in a single experiment). (d.1) Overview of Transferrin and HSA co-localization with syntaxin-4 at the basolateral membrane. Arrowheads point to two-channel overlapping pixels. Scale bar: 20 µm. (d.2) Co-localization of transferrin and HSA with the t-SNARE protein syntaxin-4. Results are expressed as mean±SEM (5 fields analyzed per experimental condition across 175-225 cells in a single experiment). Results are given by the Mander’s co-localization coefficient M1, normalized to the control. Statistical analysis was performed by a Mann–Whitney test (**: p ≤ 0.01). In b.1, b.2, d.1 and d.2, images were acquired in a LSM710 confocal microscope (Zeiss). (e.1) Representative images of sEVs, sEVs-HA, and sEVs-HA-Tf interaction with Syntaxin-4, acquired on STED mode at the plane closest to the glass slide (corresponding to the basolateral side of the cell). Scale bar: 5 µm. Inset: Higher magnification images of EVs in contact with Syntaxin-4. Scale bar: 1 µm. (e.2) Co-localization of sEVs with syntaxin-4, given by the number of sEVs carrying organelles that colocalize with Syntaxin-4. Colocalization is defined as all Syntaxin-4 objects distancing ≤200 nm from sEVs carrying organelles. Results were normalized to the control. (f.) Number of basolateral sEVs carryring organelles, normalized to the control. Results in e.2 and f. are expressed as mean±SEM (11-13 fields analyzed per experimental condition across 25-50 cells in 2 independent experiments). An independent experiment is defined as a separate bioconjugation reaction performed using sEVs obtained from 2-3 isolations derived from pooled plasma of 4-6 individual donors. Statistical analyses were performed by One-way ANOVA, followed by Tukey’s multiple comparisons test (p<0.05).

Article Snippet: To evaluate the expression of the t-SNARE protein Syntaxin-4 on the basolateral membrane, cells were seeded on type-I collagen coated ibidi μ-Slide 15 wells 3D, at a density of 50,000 cells/cm 2 , for 2 days, fixed and immunostained for Syntaxin-4 1:100 (R&D Systems, MAB7894), followed by incubation with Alexa-fluor 488 donkey anti-mouse 1:800 secondary antibody in 1% (w/v) BSA for 1 h in the dark at room temperature.

Techniques: Membrane, Expressing, Microscopy, Control, MANN-WHITNEY, Derivative Assay, Clinical Proteomics

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