Figure 5.

HOS15 Associates with EC Components.

(A) BiFC analysis of proteins transiently expressed in N. benthamiana leaves. VN and VC indicate the N and C termini, respectively, of Venus (eYFP). Fluorescence (left panels) was detected by confocal microscopy. Right panels are overlay of fluorescence and differential interference contest images. Bars = 100 μm.

(B) HOS15 interacts with ELF3. Wild type (10 d old) and hos15-2 plants were cross-linked in 1% formaldehyde to preserve in vivo interaction after harvesting plant material. Total protein extracts were immunoprecipitated with α-HOS15 antibody and resolved by SDS-PAGE. Immunoblots were probed with α-HOS15, or with α-ELF3 to detect ELF3. *Nonspecific bands.

(C) HOS15 forms a complex with EC components. N. benthamiana plants were infiltrated with Agrobacterium harboring 35S:HA-LUX, 35S:FLAG-HOS15, and 35S:MYC-ELF3 for transient expression. The proteins were immunoprecipitated with α-HA antibody and resolved by SDS-PAGE. The immunoblots were probed with α-HA to detect LUX, α-HOS15, or α-MYC to detect ELF3.

(D) Gel-filtration analysis of HOS15, LUX, and ELF3. Total proteins extracted from 2-week-old wild type plants were fractionated by size-exclusion chromatography using a Superdex 200 10/300GL column equilibrated with elution buffer [50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 0.02% sodium azide]. The samples were separated by SDS-PAGE and subjected to immunoblotting analysis using α-HOS15, α-LUX, or α-ELF3. Aliquots of total protein extracts from wild type, input after ammonium sulfate precipitation of wild type total extracts, and total protein extracts individually isolated from elf3-1, hos15-2, or pcl1-1 mutants were used as input controls. Molecular mass markers (ferritin, 440 kD; β-amylase, 200 kD; alcohol dehydrogenase, 150 kD; BSA, 67 kD) were independently eluted using the same equilibrated column. Total, total protein extract; Input, input after ammonium sulfate precipitation.