FIG 4.

FLAG-tagged Ank4_01 and Ank9 interact with ectopically expressed and endogenous SCF1 ubiquitin ligase complex components. (A) GST-tagged SCF1 ubiquitin ligase components coprecipitate FLAG-Ank4_01 and FLAG-Ank9. FLAG-tagged Ank4_01, Ank9, and BAP (negative control) were each coexpressed in HeLa cells with GST-tagged SKP1, CUL1, or RBX1. (Top row) GST pulldowns were performed, and the resulting Western blots were probed with FLAG antibody to detect coprecipitated FLAG proteins. (Middle rows) Precipitation of GST-SKP1, GST-CUL1, and GST-RBX1 was confirmed by stripping and reprobing the blots with GST antibody. The asterisk denotes a nonspecific background band that was detected by GST antibody. (Bottom row) FLAG-tagged-protein expression was confirmed using FLAG antibody to screen input samples. (B) FLAG-tagged Ank4_01 and Ank9 coprecipitate endogenous SCF1 components. FLAG-tagged Ank4_01, Ank9, and BAP were precipitated from lysates of transfected HeLa cells using FLAG affinity resin. Native SCF1 ubiquitin ligase components that coprecipitated with FLAG-tagged proteins were detected with SKP1, CUL1, and RBX1 antibodies. Expression and precipitation of FLAG-tagged proteins was confirmed using FLAG antibody. The specificity of Ank9 antiserum was verified by probing the FLAG pulldown blots to detect FLAG-Ank9 but neither FLAG-Ank4_01 nor FLAG-BAP. The data presented are representative of at least two experiments with similar results.