Fig. 8.

PhRING protein regulates PhCHS degradation in vivo. (A) PhCHS and PhRING-H2 relative expression in bud scale (mean ±SD; n=3). (B) Comparison of the abundance of ubiquitinated proteins from bud scale treated by transient transformation of PhRING-H2-OE or RNAi after injection for 72 h. PhRING-H2-OE and RNAi were performed as described in ‘Materials and methods’. Control samples were transfected with the empty vector. For the ubiquitination assay, PhCHS antibody (2 μg final concentration) was added to the extracted crude protein from bud scale and incubated with PhCHS proteins for immunoprecipitation, and the captured PhCHS proteins were assayed for ubiquitination analysis using ubiquitin antibody. Ubiquitin antibody (1:1000, from Beyotime, Shanghai, China) was used to evaluate ubiquitinated proteins. β-Actin antibody (1: 3000) was used as an internal control for evaluating the abundance of reference protein in total proteins. (C) Bud scales injected with PhRING-H2-OE or RNAi for 72 h following MG132 (40 μM final concentration) and DMSO treatment. PhCHS protein content was detected at 0 and 3 h after treatment with MG132 or DMSO. HFT, hours after treatment. PhCHS (1: 3000) antibody was used to evaluate PhCHS protein abundance in bud scales of ‘He Xie’. Control samples were transfected with the empty vector. β-Actin antibody (1: 3000) was used as an internal control for evaluating reference protein abundance in total protein content.