Extended Data Fig. 3: Interaction of NSF with αSNAP2 and impact of their dominant-negative variants on Arabidopsis seedling development.
From: NSF/αSNAP2-mediated cis-SNARE complex disassembly precedes vesicle fusion in Arabidopsis cytokinesis
(a-d) Seedling abnormalities caused by estradiol (EST)-induced expression of NSFEQ:G (a) or R:αS2LA (b) were analyzed 5 days after germination (5DAG) on EST plates. Below: Boxed areas at higher magnification. C, non-transformed wild-type; two transgenic lines (1, 2) each for the wild-type (NSF:G, R:αS2) and dominant-negative (NSFEQ:G, R:αS2LA) constructs. (c, d) Control seedlings grown in estradiol solvent DMSO for 5 days. Scale bars, 1 cm. Number of seedlings analysed, ≥200 (a-d). (e-f) Expression of transgene-encoded proteins. Protein extracts from seedlings of two transgenic lines (1, 2) incubated with EST (inducer) or DMSO (solvent control) for 24 hours were separated of SDS-PAGE gels and immunoblotted (IB) with anti-GFP (e) and anti-RFP (f) antibodies. C, non-transformed wild-type; two transgenic lines (1, 2) each for the wild-type (NSF:G in e, R:αS2 in f) and dominant-negative (NSFEQ:G in e, R:αS2LA in f) constructs. Molecular marker size on the left (kDa, kilodalton); Pon S, Ponceau S-stained membrane as loading control. (g) Co-immunoprecipitation of NSF:G and R:αS2. T, total extract; U, unbound; IP, immunoprecipitate (IP-GFP, anti-GFP beads). IB, immunoblot with antibody indicated (right); Molecular marker size on the left (kDa, kilodalton) NSF:G/R:αS2, doubly transgenic; NSF:G or R:αS2, singly transgenic.
