Figure 1.

Serines S231 and S257 of AGL15 are phosphorylated in stage 15 floral receptacles. (A) AGL15 expressed as a hemagglutinin (HA) tag in stage 15 floral receptacles, driven by its native promoter, runs a multiple bands on 25 µM Phos-tag gels.¹⁶ Mutating serines 231 and 257 to alanine reduces phosphorylated isoforms of AGL15. Exposures were chosen for each lane that scaled the 2 lanes to similar intensity for the lower, non-phosphorylated band. Expression was performed in the AGL15 null mutant, agl15-4.¹⁸ (B) Quantified ratio of phosphorylated to non-phosphorylated AGL15. Three independent lines were assayed. *P < 0.05 (t-test). n = 3; SEM error bars shown. Floral receptacles (1 mm hand cut cross section of the base of the flower) were ground in liquid nitrogen and then crude protein extracts were prepared by mixing the ground tissue with 1x SDS sample buffer (1 receptacle per 12 ul SDS sample buffer). Phos-tag gel electrophoresis and Western blotting were performed as described.¹⁶ Quantification of Western blots was performed with Image Lab software (Bio-Rad). Mutations to the previously described AGL15 native promoter construct were made via the QuikChange method (Agilent).¹⁶