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: PLoS ONE

Article Title: Intracellular Bacteria Encode Inhibitory SNARE-Like Proteins

doi: 10.1371/journal.pone.0007375

Figure Lengend Snippet: SNARE proteins description.

Article Snippet: Plasma membrane t-SNARE proteins Syntaxin3/His 6 -SNAP23, Syntaxin4/His 6 -SNAP23, Syntaxin 2/His 6 -SNAP23 were co-expressed in BL21 (DE3) star E. coli (Invitrogen) and co-purified using the His 6 tag present on SNAP23.

Techniques:

Journal: PLoS ONE

Article Title: Intracellular Bacteria Encode Inhibitory SNARE-Like Proteins

doi: 10.1371/journal.pone.0007375

Figure Lengend Snippet: C tr IncA (A) and C ca IncA (B) were reconstituted with the exocytic t-SNARE complexes [Syn2/SNAP23], [Syn3/SNAP23] and [Syn4/SNAP23]. After mixing t-SNARE liposomes (with or without IncA) with VAMP2 liposomes, fusion proceeded. Bar graphs represent the mean from n = 5 independent experiments at 30min, 60 min and 120 min for each of the exocytic complex. For the purpose of comparison, maximal values of fusion obtained for the SNARE complex without IncA at 120 min were arbitrarily defined as 100%. The standard deviation is shown. A- As shown on the curves and bar graphs, C tr IncA does not affect exocytic fusion regardless of its concentration (p>0.05). B- After 2 hrs of fusion, CcaIncA significantly inhibits [Syn2/SNAP23]-mediated fusion by 35%, [Syn3/SNAP23]-mediated fusion by 25% and [Syn4/SNAP23]-mediated fusion by 20% (p = 0.0079). One and two asterisks denote statistically significant differences with p<0.05, and p<0.02 respectively.

Article Snippet: Plasma membrane t-SNARE proteins Syntaxin3/His 6 -SNAP23, Syntaxin4/His 6 -SNAP23, Syntaxin 2/His 6 -SNAP23 were co-expressed in BL21 (DE3) star E. coli (Invitrogen) and co-purified using the His 6 tag present on SNAP23.

Techniques: Standard Deviation, Concentration Assay

Journal: PLoS ONE

Article Title: Intracellular Bacteria Encode Inhibitory SNARE-Like Proteins

doi: 10.1371/journal.pone.0007375

Figure Lengend Snippet: A- Resting transfected RBL-2H3 cells were co-labeled with anti-Myc Abs and lysotracker, and viewed by confocal microscopy. Myc-CcaIncA 1–220 /GFP is on the left, while GFP control is on the right. Co-localized Myc-CcaIncA 1–220 and lysotracker compartments are indicated with a yellow box and arrows. B-RBL-2H3 cells were transiently transfected with Myc-CcaIncA 1–220 /GFP or with GFP alone. Total lysates were migrated on SDS-PAGE and probed with Abs directed against Myc. Equivalent amounts of protein in each lane was verified after reprobing the blots with the anti-SNAP23. After stimulation of the transfectants at different time points with 10 −7 M PMA/10 −6 M ionomycin, the kinetics of degranulation was analyzed using the β-hexosaminidase release assay. The mean of triplicates from five independent experiments was determined. Standard errors are shown. For the purpose of comparison, maximal values of degranulation obtained for GFP-transfected cells at 60 min were arbitrarily defined as 100%. Transfection of Myc-C ca IncA 1–220 (Grey bars) reduces mast cells degranulation by 23% at 30 min and 31.8% at 60 min compared with GFP (Dark bars). The asterisks denote statistically significant difference (p<0.05) to GFP transfectants. Note that Myc-C ca IncA 1–220 /GFP and GFP are not statistically different at 15 min (p = 0.26).

Article Snippet: Plasma membrane t-SNARE proteins Syntaxin3/His 6 -SNAP23, Syntaxin4/His 6 -SNAP23, Syntaxin 2/His 6 -SNAP23 were co-expressed in BL21 (DE3) star E. coli (Invitrogen) and co-purified using the His 6 tag present on SNAP23.

Techniques: Transfection, Labeling, Confocal Microscopy, SDS Page, Release Assay