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. 2018 Mar 30;35(1):1–7. doi: 10.5511/plantbiotechnology.17.1208a

Figure 3. Phosphatase inhibitors treatment induced the phosphorylation of AtHMGR1S at sites other than S577. Transgenic A. thaliana seedlings (14 day old) were treated with phosphatase inhibitors cocktail and incubated for an additional 6 h. The seedlings were then used for FLAG-tag protein purification. FLAG-tag purified proteins were then fractionated with Phos-tag SDS-PAGE, followed by western blotting analysis using an anti-HMG1cd antibody. The phosphorylated HMGR has slower electrophoretic mobility (indicated by black arrow and asterisks) compared to the non-phosphorylated HMGR (indicated by blank arrow) in Phos-tag SDS-PAGE. There are three additional bands from AtHMGR1S (black arrow-1, 2, 3) and one additional band from AtHMGR1S_S577A FLAG-tag purified proteins (black arrow-2) from the seedlings treated with phosphatase inhibitors. The lambda phosphatase treatment abolished the additional bands of the FLAG-purified proteins. The Phos-tag SDS-PAGE of samples in lanes 1, 2, 10, and 11 was repeated without the addition of lambda phosphatase buffer to get sharper separation of protein bands and shown in Supplementary Figure S2.

Figure 3. Phosphatase inhibitors treatment induced the phosphorylation of AtHMGR1S at sites other than S577. Transgenic A. thaliana seedlings (14 day old) were treated with phosphatase inhibitors cocktail and incubated for an additional 6 h. The seedlings were then used for FLAG-tag protein purification. FLAG-tag purified proteins were then fractionated with Phos-tag SDS-PAGE, followed by western blotting analysis using an anti-HMG1cd antibody. The phosphorylated HMGR has slower electrophoretic mobility (indicated by black arrow and asterisks) compared to the non-phosphorylated HMGR (indicated by blank arrow) in Phos-tag SDS-PAGE. There are three additional bands from AtHMGR1S (black arrow-1, 2, 3) and one additional band from AtHMGR1S_S577A FLAG-tag purified proteins (black arrow-2) from the seedlings treated with phosphatase inhibitors. The lambda phosphatase treatment abolished the additional bands of the FLAG-purified proteins. The Phos-tag SDS-PAGE of samples in lanes 1, 2, 10, and 11 was repeated without the addition of lambda phosphatase buffer to get sharper separation of protein bands and shown in Supplementary Figure S2.