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. 2016 May 3;18(11):1596–1610. doi: 10.1111/cmi.12599

Figure 2.

Figure 2

Palmitoyl‐transferase activity assay demonstrating the palmitoyl‐transferase activity of the PfDHHC proteins on PfSec22. A. Immunoprecipitation of PfSec22 co‐expressed with each PfDHHC protein. Human embryonic kidney 293E cells were co‐transfected with plasmids coding for the expression of c‐Myc‐tagged PfSec22, along with the indicated FLAG‐tagged PfDHHC proteins (PfDHHC3, 5, 7 and 9) or the control vector (CD4). Pfsec22 was immunoprecipitated from cell lysates using α‐c‐Myc antibody. The proteins were separated by SDS‐PAGE and visualized by immunoblot, using α‐c‐Myc antibody from a different species. B. PAT activity assay. Human embryonic kidney 293E cells were co‐transfected with plasmids expressing c‐Myc‐tagged PfSec22, along with the indicated FLAG‐tagged PfDHHC protein or the control vector (CD4). Cells were either treated with the metabolic label, 17‐octadecynoic acid (17‐ODYA) or mock‐treated with DMSO. Proteins were extracted and an aliquot of each lysate kept aside to confirm protein expression. The remaining lysates were put through click chemistry reactions to biotin‐azide, and 17‐ODYA‐labelled proteins were streptavidin affinity purified and eluted by boiling in SDS. Samples from the initial lysates and the click chemistry elutions were separated by SDS‐PAGE, and the presence of c‐Myc‐tagged PfSec22 in each of the samples was observed by immunoblot using antibodies against the c‐Myc tag.