Figure 2. Interaction analysis of cytokinetic SNAREs with traffic blocked at the TGN.

Wild-type (WT) and big3 mutant seedlings carrying estradiol-inducible YFP:NPSN11 (A) or GFP:SNAP33 (B) transgenes were treated with 50 µM BFA for 30 min followed by 50 µM BFA + 20 µM estradiol for 210 min (see Figure 1B–C). Protein extracts were subjected to immunoprecipitation with anti-GFP beads, protein blots were probed with the antisera indicated on the right (IB): GFP, anti-GFP; KN, anti-KNOLLE; V721/V722, anti-VAMP721/722; SYP71, anti-SYP71; SNAP33, anti-SNAP33; kDa, protein size (left); MW, molecular weight; -BFA, mock treatment; +BFA, BFA treatment; T, total extract; UB, unbound; IP, immunoprecipitate. Loading (%), relative loading volume to total volume; relative signal intensity (input signal = 100% for UB and IP). The experiments were technically repeated more than six times.

DOI: http://dx.doi.org/10.7554/eLife.25327.006

Figure 2—figure supplement 1. Control experiments for co-immunoprecipitation analysis of cytokinetic SNAREs.

(A) No EST-induction of expression of KNOLLE SNARE partners. big3 mutant seedlings carrying YFP:NPSN11 (left) or GFP:SNAP33 (right) transgenes were treated or not treated with 50 µM BFA for 240 min. Seedlings treated with 20 µM estradiol and 50 µM BFA for 210 min were used as positive control. Protein extracts were subjected to immunoprecipitation with anti-GFP beads, protein blots were probed with the antisera indicated on the right (IB). (B) In vitro mixing of experimental extract with different amounts of extract from tagged KNOLLE seedlings. Cleared protein extracts of YFP:N11 or GFP:SNAP33 and the cleared protein extracts of mCherry:KNOLLE were mixed and subjected to immunoprecipitation with anti-GFP beads. Protein blots were probed with the antisera indicated on the right (IB). Note that the addition of mCherry:KNOLLE protein does not alter the amount of endogenous KNOLLE co-immunoprecipitated with YFP:N11 or GFP:SNAP33. Non, no addition of mCherry:KNOLLE protein lysate; 1x, equal amounts; 10x, 10x excess. (C) Seedlings of wild-type (WT) background carrying YFP:NPSN11 or GFP:SNAP33 transgenes were treated with 50 µM BFA + 20 µM estradiol for 210 min. Protein extracts were subjected to immunoprecipitation with anti-GFP beads, protein blots were probed with the antisera indicated on the right (IB). GFP, anti-GFP; RFP, anti-RFP; KN, anti-KNOLLE; V721/V722, anti-VAMP721/722; kDa, protein size (left); MW, molecular weight; -EST, no estradiol; -BFA, mock treatment; +BFA, BFA treatment; T, total extract; UB, unbound; IP, immunoprecipitate; Loading (%), relative loading volume to total volume; relative signal intensity (input signal = 100% for UB and IP).

DOI: http://dx.doi.org/10.7554/eLife.25327.007