Transcript Supplementary Figure 3 - PowerPoint (474 KB )

Supplemental 3: Specificity of anti-Skp1 and anti-cullin antibodies against C. elegans
homologs
b
30
20
SKR-2
IB: AntiSkp1
kDa
200
IB: AntiCullin
126
NS
Could be NS
96
SKR-1
SKR-3/4
-tubulin
RNAi
cul-1
skr-2
kDa
56
38
none
skr-1
RNAi
none
a
or Cul2/4
CUL-1
56
IB: anti-tubulin
-tubulin
IB: anti-tubulin
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.