Journal: Communications Biology

Article Title: Dynamic palmitoylation events following T-cell receptor signaling

doi: 10.1038/s42003-020-1063-5

Figure Lengend Snippet: In vitro palmitoylation of VAMP7 by DHHC18 and DHHC20. a Schematic cartoon of constructs used, with important cysteines highlighted. Cys222 and Cys15 are the active sites of DHHC18 and DHHC20, respectively. Cys183 in VAMP7 is the site of palmitoylation. b Cartoon model of potential membrane docking of DHHC18 isoform 2 via Cys138. The full-length DHHC18 features the two N-terminal TMDs, securing the DHHC active site to the membrane. The short isoform, lacking these TMDs, may be anchored to the membrane via palmitoylation of Cys138, allowing its active-site access to the membrane environment required for its activity. c Schematic representation of the on-plate palmitoylation assay (OPPA). Briefly, the target (VAMP7, featuring both His6 and FLAG-tags) is bound to a Ni-NTA plate. The DHHC PAT is added with clickable PAA-CoA and the reaction is quenched at specific time points using NEM. Biotin is subsequently clicked on to the PAA-modified target, VAMP7 is eluted and then bound to an anti-FLAG plate. Streptavidin-Eu3+ can then be used to quantify palmitoylated VAMP7, and a steady-state slope can be calculated. d Results of OPPA experiments of VAMP7-WT and VAMP7-C183A, palmitoylated by constructs of DHHC18 and DHHC20 ( n ≥ 12, geometric mean ± SEM shown).

Article Snippet: SILAC-labeled heavy and light samples were then mixed and allowed to bind to streptavidin-agarose beads (Novagen) for 90 min at room temperature.

Techniques: In Vitro, Construct, Activity Assay, Modification

Journal: Communications Biology

Article Title: Dynamic palmitoylation events following T-cell receptor signaling

doi: 10.1038/s42003-020-1063-5

Figure Lengend Snippet: Enrichment of Jurkat T-cell palmitome before and after anti-CD28/anti-CD3-costimulation by acyl-biotin exchange. a Schematic representation of acyl-biotin exchange (ABE) protocol coupled with SILAC for quantitation of palmitoylated proteins via LC-MS/MS. Briefly, Jurkat cells grown in heavy SILAC media (blue) were lysed and free thiols were blocked with NEM. After cleaving palmitoyl groups with HA, previously palmitoylated thiols are biotinylated with HPDP-biotin, allowing for streptavidin enrichment. For control cells grown in light SILAC media (red), HA is omitted, preventing biotinylation. Proteins quantified with heavy/light intensity ratios > 2.0 are considered enriched and palmitoylated. b Volcano plot showing high-confidence palmitoylated proteins prior to stimulation ( n = 6, p

Article Snippet: SILAC-labeled heavy and light samples were then mixed and allowed to bind to streptavidin-agarose beads (Novagen) for 90 min at room temperature.

Techniques: Quantitation Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Scientific Reports

Article Title: Vesicular stomatitis virus G protein transmembrane region is crucial for the hemi-fusion to full fusion transition

doi: 10.1038/s41598-018-28868-y

Figure Lengend Snippet: Replacement of the VSV G TM region with TM regions from other membrane fusion proteins resulted in similar protein expression levels and plasma membrane localization. ( a ) Sequences of the TM regions of VSV G, Syntaxin 1 A, VAMP2 and influenza HA. ( b and c ) VSV G TM replacement did not lead to dramatic changes in the expression levels of VSV G in HeLa cells. ( d ) WT and chimeric VSV G localized onto the plasma membrane. HeLa cells were transfected with plasmids encoding WT or chimeric VSV G proteins fused with EGFP. ( e and f ) Biotinylation assays to quantify membrane-localized WT or chimeric VSV G proteins. Total VSV G levels in cells (I: input) and membrane-localized G protein levels (P: pull down by streptavidin resin) were determined by western blot. The ratio of biotinylated VSV G to total G protein was calculated. Compared to WT VSV G, chimeric mutants showed similar levels of membrane localization. Mean ± SEM.

Article Snippet: Biotinylated membrane proteins were pulled down by streptavidin agarose resin (Thermo fisher scientific, 20349) following the manufacturer’s instructions.

Techniques: Expressing, Transfection, Western Blot