CLCuMuB βC1 interferes with the interaction between NbCUL1 and NbSKP1.1 in vitro and in vivo.

(A) GFP competitive pull-down assay in vitro. His-βC1 was expressed in E. coli as inclusion body and refolded through urea-arginine dialysis. BSA (NEB, USA) was used as a control. GFP-NbCUL1 or GFP was expressed in N. benthamiana leaves and trapped through GFP-Trap agarose. After the supernatant was discarded, GFP-Trap agarose was incubated with E. coli-expressed His-HA-NbSKP1.1, then the supernatant was discarded. GFP-Trap agarose was incubated with gradient dilutions (1, 1/2, 1/4) of His-βC1. Finally, agarose was washed and proteins were analyzed via SDS-PAGE and western blot assays using anti-GFP and anti-HA antibodies. Input was analyzed by the anti-His antibody (EASYBIO, China) and supernatant was analyzed by the anti-HA antibody. Intensity was detected through Total Lab TL120. (B) A confocal image of BiFC assays show that CLCuMuB βC1 interfered with the interaction between NbCUL1 and NbSKP1.1 in vivo. Photos were taken at 48 hpi. Bar scale represents 200 μm. (C) BiFC intensity (means±SEM, n = 4) was quantified by YFP fluorescence. Relative BiFC intensity was normalized to the control. The raw data were analyzed by two-sample t-test to show the significance level at 0.01 (**). (D) The protein level of cYFP-NbCUL1 and nYFP-NbSKP1.1 were checked with the polyclonal GFP antibody (Huaxin Bochuang, China). The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as the loading control.