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. 2016 Aug 15;14(8):e1002534. doi: 10.1371/journal.pbio.1002534

Fig 3. Physical interaction of Sec4p with actin patch subunits.

Fig 3

A. Top panel: representative in vitro binding assay showing in vitro transcribed and translated 35S-Las17p binding to bacterially expressed GST-Sec4p purified and immobilized on beads prior to SDS-PAGE and autoradiography. Average percentage of input Las17p interacting with GDP- or GTPγS-bound GST-Sec4p as shown (n = 3). Bottom panel: SDS-PAG showing equal amounts (1 μg) of GST-Sec4p, GST, and GST-Ypt1p preloaded with GDP or GTPγS prior to 35S-Las17p addition. B. BiFC assays for cells expressing Sla2p-YFPN (CBY4625) or YFPN-Sec4p (CBY4629) when mated with cells expressing Las17p-YFPC (CBY4660), Sla2p-YFPC (CBY4661), or Abp1p-YFPC (CBY4632). Fluorescence at the cell cortex (arrowheads) indicates in vivo interactions at actin patches (bar = 5 μm). C. Competition of BiFC binding following overnight PGAL-LAS17 induction or 6 h PGAL-SEC4 induction with galactose (Gal), compared to no induction in glucose (Glc) medium, in cells expressing YFPN-Sec4p and Las17p-YFPC (CBY4638). In all cells observed (including controls), non-specific cytoplasmic fluorescence increased after transfer to galactose-containing medium. D. Bar graphs quantifying reductions in BiFC particles within cells corresponding to images shown in panels B and C (n > 100 cells).