Article Title: CHLOROPLAST UNUSUAL POSITIONING 1 is a new type of actin nucleation factor in plants
Figure Lengend Snippet: Effects of mutations at R820 and F958 of CHUP1-C on the activity of CHUP1 (A) A schematic illustration of CHUP1 protein showing the functional domains: the hydrophobic region, the coiled-coil domain, the F-actin binding domain, the proline-rich FH1-like domain, and the FH2-like domain. Asterisks indicate R820, which is involved in the association with actin, and F958, which is involved in the dimerization of the FH2-like domain. The positions of the 611–1004 fragment (CHUP1-C) and the 611–982 and 716–982 deletion mutations are indicated by lines below the structure. (B–C) The effect of point mutations R820D and F958D on CHUP1-C dimer structure. (B) The elution profiles of CHUP1-C_WT, R820D, and F958D recombinant proteins were obtained by gel filtration chromatography using a Superdex 200 10/300 column. (C) The protein profiles were confirmed using 10% SDS-PAGE gels. (D–E) Ultracentrifugation assay of actin polymerization. ACT7 (4 µM) was allowed to polymerize in F-buffer containing 8 µM PRF1 and 0.4 µM WT or mutant CHUP1-C. (D) A representative SDS-PAGE gel of supernatant (S) and pellet (P) fractions after ultracentrifugation. (E) A bar graph showing the fraction of ACT7 in the pellet and the supernatant under each condition, with error bars showing SD (N = 3). (F) Immunoblot analysis showing the expression levels of CHUP1-YFP_R820D and CHUP1-YFP_F985D in C1Y_R820D (18-2 and 26-3) and C1Y_F958D (6-5 and 10-1) transgenic plants. The arrowhead and arrow indicate CHUP1 and CHUP1-Y, respectively. * indicates truncated CHUP1 and CHUP1-Y. The details are the same as in Figure 2A . (G) Chloroplast photorelocation movement in WT, chup1 mutant ( chup1-3 ), and C1Y_R820D and C1Y_F985D transgenic plants in a chup1 background. The red light (RL) transmittance in rosette leaves was monitored under different intensities of blue light (BL; 0, 2.8, and 50 µmol m −2 s −1 ) for the indicated periods.
Article Snippet: The eluted CHUP1-C was further purified in a buffer (10 mM HEPES pH 7.4, 100 mM NaCl, 2 mM MgCl2 , 1 mM DTT) by size-exclusion chromatography using a Superdex 200 10/300 column (GE Healthcare).
Techniques: Activity Assay, Functional Assay, Binding Assay, Recombinant, Filtration, Chromatography, SDS Page, Mutagenesis, Expressing, Transgenic Assay