Fig. 2.

Subcellular localization of the MKKK18 protein in Arabidopsis protoplasts. Active MKKK18 is localized in the nucleus. (A) Microscopy images show nuclear localization of the MKKK18–GFP fusions in Arabidopsis protoplasts. Hoechst H33342 was used as nuclear localization marker. Scale bars are calibrated to 20 µm. (B) MKKK18 protein expression and activity were analyzed by immunoprecipitation using protein-specific anti-MKKK18 antibodies and immunocomplex kinase assay with MBP as a substrate. The immunocomplex assay confirmed residual kinase activity of the MKKK18K32M-GFP allele. Western blot detection using anti-GFP antibodies corroborates the presence of MKKK18–GFP fusion proteins. Coomassie Brilliant Blue (CBB) staining confirmed equal loading.