Figure 2.

AGL15 phosphomimetics have similar mobility shifts as 35S-AGL15 from floral receptacles. AGL15 produced by the 35S promoter in floral receptacles runs as 2 bands on one day old 12% SDS-PAGE gels. The slower mobility band can be shifted to the faster mobility band by phosphatase treatment.¹⁶ However, 35S-AGL15 expressed in mesophyll protoplasts runs as a single band. Mutating either S231 or S257 to aspartate reduces AGL15 mobility with the double substitution causing the biggest mobility shift similar to phosphorylated AGL15. The experiment was repeated twice with the same result. Protoplast preparation and transfection was performed as described.¹⁹ Six hours after transfection, protoplasts were solubilized in SDS sample buffer. SDS-PAGE and Western blotting were performed as described.¹⁶ 35S-AGL15 floral stage 15 receptacle extract was prepare as described in the Fig. 1 legend. cDNA from the previously described construct used to generate 35S-AGL15-double HA tagged plants was inserted into pRLT2 for protoplast studies and mutations were made via the QuikChange method (Agilent).¹⁶