FIGURE 5.

AXR1 neddylation does not affect AXR1 stability or apparent protein interactions in vivo. A, IB analyses with anti-Myc, anti-RGA, and anti-CDC2 following a CHX chase experiment with wild type (WT) seedlings and axr-30 or axr-30 den1-1 mutant seedlings expressing Myc:AXR1. Seedlings were grown on growth medium for 7 days and subsequently transferred to liquid growth medium with 50 μm CHX for up to 6 h. The anti-CDC2 IB serves as a loading control; the anti-RGA IB against the unstable RGA protein serves as a positive control for the CHX treatment. B, graph depicting the normalized signal intensities of Myc:AXR1 in its unmodified (AXR1) and NEDD8-modified form (AXR1-N8) as detected in axr-30 or axr-30 den1-1 backgrounds. In the case of the axr-30 den1-1 measurements, the results from AXR1 and AXR1-N8 were summed up to provide a measure for the overall AXR1 abundance. C, IB with anti-Myc of 20 μl eluate per fraction following a Superose 6 gel filtration of total protein extract from 7-day-old Myc:AXR1 axr-30 den1-1 seedlings. Fraction numbers and corresponding molecular weights are specified accordingly. N8, NEDD8.