(A) Solubilization of proteins from RPs. Proteins on RPs were solubilized by stepwise treatment with buffers containing 4 mM CHAPS (lane 1), 16 mM CHAPS (lane 2), and 7 M urea, 4 M thiourea, and 6.5 mM CHAPS (lane 3), analyzed using SDS-PAGE, and then visualized by silver staining. (B) Relative RTase activities of the RPs, with RTase activity of non-detergent-washed RPs set at 1.0. 1: Sustained activity on RPs after the second wash with 16 mM CHAPS; 2: activity of the second-washed RPs co-incubated with the proteins released by the two-step washes, corresponding to the mixture of samples shown in Figure 2A, lanes 1 and 2, after dialysis removal of the detergent. RTase activities were assayed using the standard conditions (see Materials and methods). Results are presented as the mean of three independent determinations ± SD. (C) Molecular phylogenetic tree of the amino acid sequences of identified NgBR family proteins. The tree was constructed using the neighbor-joining method and drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Tree members are HRBP, the NgBR homologue from H. brasiliensis (LC057267); LsCPTL1 from L. sativa (AIQ81186); LsCPTL2 from L. sativa (AIQ81187); SlCPTBP from Solanum lycopersicum (XP_004241992); LEW1 from A. thaliana (NP_001077518); Nus1p from S. cerevisiae (NP_010088); NgBR (AAI50655), and non-functionally identified NgBR-like proteins in Oryza sativa (Os02g0197700: NP_001046201, Os03g0197000: NP_001049268) and Glycine max (Glyma01G233700: NP_001242452, Glyma11G009100: XP_003537718). (D) Tissue specificity of the expression levels of the NgBR homologue from H. brasiliensis. Transcript levels were determined as copy number of HRBP, normalized with those of the 18S rRNA quantified from the total RNA extracted from latex, leaves, stems, roots and suspension-cultured cells of H. brasiliensis. Results are presented as the means of three independent determinations ± SD. RP, rubber particle.