IB: anti-  -tubulin

1. IB: anti- -tubulin IB: anti- -tubulin -tubulin -tubulin Supplemental 3: Specificity of anti-Skp1 and anti-cullin antibodies against C. elegans homologs a b RNAi RNAi IB: Anti- Cullin cul-1 none skr-1 none skr-2 IB: Anti-Skp1 kDa kDa 56 200 38 126 NS Could be NS or Cul2/4 30 96 SKR-1 20 CUL-1 SKR-2 SKR-3/4 56 Specificity of anti-Skp1 and anti-cullin antibodies against C. elegans homologs. RNAi experiments were performed by treating N2 (wild-type) animals with bacteria clones expressing dsRNA against SKR-1, SKR-2 and CUL-1. Lysates were analyzed using antibodies against Skp1 (a) and cullin (b), in addition to anti--tubulin (bottom panels) for loading control. Epitope for anti-Skp1 antibody shares higher homology with SKR-1(20 kDa) than SKR-2(19.5 kDa), and SKR-2 may be detected occasionally. SKR-3/4 may be detected weakly (18 and 16 kDa) as they contains only weak homology to the antibody recognition site. The band at 30 kDa is likely non-specific (NS). The anti-cullin antibody recognizes a band of with the predicted molecular weight of CUL-1 (87.5 kDa) and this band is significantly reduced with treatment of dsRNA against cul-1. Other bands could be non-specific (NS) or Cul2/4. Methods: We used RNAi to knockdown specific SKRs and CULs in C. elegans because the genetic mutants are either not generated or homozygously lethal. For RNAi treatment, bacteria harboring dsRNA expression plasmids for SKR-1(F46A9.5), SKR-2(F46A9.4) and CUL-1(D2045.6) were obtained from MRC GeneService, UK. Log-phase bacteria seeded onto NGM plate containing 100 g/mL carbanicillin and 0.5 mM IPTG. dsRNA expression was induced by incubation overnight at room temperature. Late larval stage N2 worms were placed on RNAi plates at 22oC for 3 days before the lysates were prepared for western blotting analysis.