Monoclonal antibody recognizing eyfp

Click chemistry palmitoylation assay was conducted imitating the method described by Coleman DT, et al. [15 (link)]. In brief, after the metabolic labeling the palmitoylated proteins with palmitate orthologs alkyne-linked 17-Odya (Cayman Chemical, USA), the cells lysates were further processed through the click reaction between biotin-azide (Cayman Chemical, USA) and alkyne-linked 17-Odya, which make the palmitoylated proteins covalently cross-linked with biotin at the S-palmitoylation sites. The biotinylated protein were pulled down from the cells lysates using streptavidin-agarose mini-column (Sigma, USA), and then were subjected to western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China).

The acyl-biotin exchange assay was conducted imitating the method described by Zhang Y, et al. [14 ]. In brief, the VPAC1-CHO and VPAC1-Cys37/Ala-CHO cell lysates were incubated overnight with 1M NEM (Sigma, USA) at 4°C to block all the free sulfhydryls. Fresh 1M HA (Sigma, USA) was added and the culture lasted for 1 h at room temperature to cut off the thioester bond of palmitoylation to produce extra free sulfhydryls. And 4M biotin-HPDP (ProteoChem, USA) was added later to react with the free sulfhydryls produced by HA to achieve the biotin exchange labeling of the palmitoylation. Then streptavidin-agarose mini-column (Sigma, USA) were used to gather the biotin-labeling proteins, and the final elution from the streptavidin-agarose mini-column was submitted to 10% SDS-PAGE and western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China). The treatment without HA was used as systemic control.