Extended Data Fig. 9: Semi-quantitative analysis of coimmunoprecipitation assay, interaction of NSF and KNOLLE and chromatography analysis of NSF and KNOLLE complexes.
From: NSF/αSNAP2-mediated cis-SNARE complex disassembly precedes vesicle fusion in Arabidopsis cytokinesis
(a-b) Group box plots of semi-quantitative analysis. Signal intensities of the immunoprecipitate (IP) fractions with anti-GFP (a) and anti-RFP (b) beads were measured from two or three immunoblots probed with SNARE antisera as in Fig. 3a,b: KNOLLE, NPSN11, SYP33, SYP71, VAMP721/722 (VAMP721). Band intensity values were normalized with wild type NSF and αSNAP2 set at 100; A.U, arbitrary units. For simplicity, immunoprecipitate from the non-transformed wild type extract was not included. The P values were calculated using unpaired Student’s t-test (two-tailed) by comparing the respective wild type to its dominant negative mutant. The center line of the whisker plots is the median, the bottom and top lines represent the lower quartile and upper quartile, respectively. n, number of the counted immunoblots. (c-d) Coimmunoprecipitation of wild type NSF:G or dominant-negative NSFEQ:G with Myc:KNOLLE (Myc:KN). Extracts from non-transformed wild-type (C) or transgenic seedlings coexpressing Myc:KN and NSF:G or NSFEQ:G were immunoprecipitated (IP) with anti-Myc beads. Protein blots were immunoblotted (IB) with antibodies indicated on the right. Myc:KN, immunoprecipitate of extract from transgenic seedlings expressing only Myc:KNOLLE. Band intensity values were normalized with wild type NSF:G in IP fraction set at 100%. Molecular marker size on the left (kDa, kilodalton); Total, total extract; IP, immunoprecipitate. (d) Semi-quantitative analysis of (c). Signal intensities of the immunoprecipitate (IP) were measured from two immunoblots. Band intensity values were normalized with wild type NSF:G in IP fraction set at 100; A.U, arbitrary units. For simplicity, immunoprecipitate from the non-transformed wild type extract was not included. The center line of the box plots is the median, the bottom and top lines represent the lower quartile and upper quartile, respectively. n, number of the counted immunoblots. Note that P value was not calculated (n.c) for n≤2. (e) Immunoblots from size-exclusion chromatography of NSF and KNOLLE complexes shown in Fig. 3c. Continuous fractions of NSF:G and NSFEQ:G in the high molecular mass range were immunoblotted (IB) with the indicated antisera anti-GFP, anti-KNOLLE (KN), and anti-VAMP721/722 (V721). INPUT, filtered protein extracts before running size exclusion chromatography; C, non-transformed wild-type protein extracts. Peaks of the standard markers are indicated (669 kDa, thyroglobulin; 443 kDa, ferritin).
