Cell, Volume 128 Supplemental Data The RSA Complex Targets Protein Phosphatase 2A to Centrosomes and Regulates Mitotic Spindle Assembly in C. elegans

independent mutant lines; 700 (left) and 800 nm (right) emission images showing the fragments generated by Cel-1-based heteroduplex cleavage. (Arrowhead) Full-length product, 790 bp; (hexagons) Cel-1 cleavage products (lane 37), which appears in both channels with complementary sizes (630 and 160 bp). After sequencing, this signal was confirmed to be a point mutation, which was the allele dd20. (B) Mutation spectrum in rsa-1. In total, 23 mutations were found, among which 8 were silent and 7 were intronic. The table only depicts the mutations affecting exons or splice sites. Mel, maternal effect lethal phenotype. (C) Genomic organization of RSA-1 and RSA-1dd13. Shown are the positions of 3 mutations (dd10, dd11 and dd13) potentially leading to a 3’-truncated structure. In dd13, the mutation affects the highly conserved donor site in the last intron, which can no longer be spliced. As a result, the RSA-1dd13 predicted protein has 12 additional residues (boxed in red) and lacks all the 69 amino acids of the last exon. (D) Western blot of whole worm extracts of wild-type, heterozygous (dd10/hT2; dd13/hT2) and homozygous mutants (dd10, dd11, dd13). RSA-1 is the upper band (arrow), detected by an antibody against the extreme C-terminus of the protein (see Experimental procedures). The lower band, which is non-specific, serves as a loading control. Two mutant forms are no longer recognized, dd10 and dd13, which confirms their predicted 3’-truncated form. A faint band is seen in dd11 animals, which could indicate stochastic splicing. Note that a Mel phenotype is associated with these 3 homozygous mutant alleles