Figure 1 An in vitro phosphorylation assay of 3 putative substrate proteins by MPK6 in Arabidopsis thaliana MPK6 was fused with the 6×His tag and the 3 putative substrates were fused with the Flag tag. After phosphorylation reaction, the proteins in the mixture were separated on a SDS- PAGE gel. The gel was dried and exposed to X-ray film (top). Immunoblotting with anti-His and anti-Flag antibodies were used to show the levels of the substrate (middle) and kinase (bottom) proteins in the reactions.

Figure 2 Immunoblotting detection of phospho-proteins in samples after Phos-tag gel and SDS-PAGE gel separation Phospho-proteins can be dephosphorylated by phosphatases (e.g. Calf intestine alkaline phosphatase, CIAP). Protein samples treated with (+) or without (-) CIAP were separated by Phos-tag (top) and SDS-PAGE (bottom) gels, and the specific protein was further detected by immunoblotting. The P-form and None-P-form of the protein were separated clearly by a Phos-tag gel (top), while the two forms were not separated by a SDS-PAGE gel (bottom). The missing of the upper band and the increasing of the bottom band after Phos-tag gel separation indicated that P-form protein was completely dephosphorylated by CIAP treatment.

Figure 3 Pro-Q staining assay of the phospho-proteins in total proteins extracted from Arabidopsis thaliana seedlings and separated by a 2D gel Two-week-old Arabidopsis thaliana seedlings were used for total protein extraction. The total proteins were separated by a 2D gel and the phospho-proteins were stained by Pro-Q (left). The gel was scanned with a Typhoon 9410 fluorescence scanner, and then stained with blue-silver to visualize the proteins (right).