MOESM2 of Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry

Patrick Boyle, Simon Schwizer, Sarah Hind, Christine Kraus, Susana Torre Diaz, Bin He, Gregory Martin
2016 Figshare  
Additional file 2: Figure S2. Alkyne fatty acid analogs do not interfere with immunity mechanisms and are incorporated in fatty acylated proteins in the context of intact plant leaf tissue. (A) Nicotiana benthamiana leaves were infiltrated with Agrobacterium strains carrying Pto and empty vector (EV) or avrPto. 50 μM Alk12, Alk14, Alk16, or buffer were infiltrated 24 h after Agrobacterium infiltration. Plants were monitored for programmed cell death and pictures taken 2 days after
more » ... after transformation. (B) Nicotiana benthamiana was used to transiently express HA epitope-tagged avrPto. 10 μM Alk12, Alk14, Alk16, or buffer was infiltrated twice, 24 h after Agrobacterium infiltration and 6 h before sampling. Tissue was collected 48 h after transformation, total protein extracted, AvrPto immunoprecipitated using anti-HA resin, and a fluorescent tag added using click chemistry. Incorporated alkyne fatty acid analogs were visualized by fluorescence imaging and total protein was detected by anti-HA western blotting. Black dividing lines indicate removal of irrelevant lanes from the blot and gel images.
doi:10.6084/m9.figshare.c.3603188_d5 fatcat:iwgjr4tf7fd4remz23v3tohdri