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. 2015 Apr 14;27(4):1098–1112. doi: 10.1105/tpc.15.00178

Figure 8.

Figure 8.

Blue Light-Induced Alteration of NPH3 Proteins in the rpt2 Mutant.

After light treatments, soluble proteins (soluble) and crude microsomal proteins (membrane) were prepared from the shoots of dark-grown seedlings. Proteins (6 μg) in each fraction were separated on 7.5% SDS-PAGE gels, followed by immunoblotting with anti-NPH3 antibody. The closed and open arrowheads indicate the positions of slightly and excessively dephosphorylated NPH3 proteins, respectively.

(A) and (B) Effects of red light (RL) pretreatment. Dark-grown seedlings were pretreated with or without red light and irradiated with continuous unilateral blue light (BL) at 0.0017 μmol m–2 s–1 (A) or 0.17 μmol m–2 s–1 (B) for the indicated periods.

(C) and (D) Effects of blue light on the phosphorylation status of phot1 and NPH3 proteins in the rpt2-1 mutant (C) and rpt2-2 transgenic plants harboring 35Spro:RPT2 (D). Dark-grown seedlings were irradiated with continuous unilateral blue light at 0.17 μmol m–2 s–1 for the indicated periods.

(E) Effects of light treatments on the interaction of NPH3 with phot1 and RPT2 in vivo. Dark-grown transgenic nph3 plants harboring 35Spro:YFP-NPH3 were irradiated with continuous unilateral blue light at 0.17 μmol m–2 s–1 for the indicated periods. The seedlings were harvested and used to prepare crude microsomal proteins. The crude extracts were immunoprecipitated (IP) with GFP agarose beads and separated on 7.5% SDS-PAGE gels, followed by immunoblotting with anti-PHOT1, anti-RPT2, and anti-GFP antibodies. A 1:200 dilution of crude microsomal proteins used for immunoprecipitation was also electrophoresed as a control for phot1 and RPT2 proteins.

(F) Quantification of the interaction of NPH3 with phot1 and RPT2. The immunoprecipitated samples (see above) were measured with ImageJ (version 1.48; http://imageJ.nih.gov/ij). The data shown are means ± se from four experiments using biologically independent samples. Asterisks indicate statistically significant differences compared with time 0 (Student’s t test, P < 0.05).