Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - No net FRET is seen with Cul3 and actin. (A) HEK293 cells expressing CFP-actin (a-e), YFP-Cul3 (f-j), and CFP-actin with YFP-Cul3 (k-o) were imaged for quantitative FRET analysis. Phase contrast (a, f and k), CFP (b, g, and l), YFP (c, h, and m), and FRET fluorescence images (d, i, and n) were acquired and net FRET values (e, j, and o) were determined using the Metamorph application.
• Supplemental Figure 2 - Rbx1 does not mediate the formation of Cul3 heterodimer. FLAG-tagged Cul3 expressed in HEK293 cells was immunoblotted with anti-FLAG antibody (bottom panel). Cell lysates were subjected to immunoprecipitation with anti-FLAG (lane 1) or anti-Rbx1 antibodies (lane 2) followed by immunoblotting with anti-FLAG antibody (top panel).
• Supplemental Figure 3 - FLAG-tagged Cul3K712R and FLAG-tagged Cul3patch A patch B mutants cannot be neddylated. HEK293 cells were transfected with vectors expressing FLAG-tagged Cul3, FLAG-tagged Cul3K712R, or FLAG-tagged Cul3patch A patch B. Lysates were prepared, checked for protein expression, and immunoprecipitated with anti-FLAG antibody. The precipitates were separated by SDS-PAGE and immunoblotted with anti-Nedd8 antibody (top panel) and anti-FLAG antibody (bottom panel).
• Supplemental Figure 4 - Cul1-Cul3 interactions are detected in vivo. HEK293 cells were transfected with vectors expressing FLAG-tagged Cul3 and HA-tagged Cul1 as indicated. Lysates were prepared, checked for protein expression (bottom two panels), and immunoprecipitated with anti-FLAG antibody. The precipitates were separated by SDS-PAGE and immunoblotted with anti-HA antibody (top panel).