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Figure S1. Verification of the specificity of UNC-98 antibodies and demonstration that UNC-98 copurifies with thick filaments. (A) Anti-UNC-98 antibodies affinity purified to the N terminus of UNC-98 more specifically recognize the UNC-98 protein. The left three lanes show reaction of UNC-98 antibodies affinity purified to full-length UNC-98 protein, as reported in Mercer et al. (2003). The right three lanes show reaction to UNC-98 antibodies affinity purified to the N-terminal 112 residues. In each case, the three lanes represent extracts from wild-type (wt) and two unc-98 mutant alleles, sf19 and su130, described in Mercer et al. (2003). Note that these later antibodies more specifically react with a 37-kD protein of expected size for UNC-98. unc-98(sf19) shows a truncated protein of reduced abundance, whereas unc-98(su130) shows a protein of wild-type size and abundance. (B) Thick filament purification, as shown in the schematic diagram (adapted from Epstein et al., 1988; Deitiker and Epstein, 1993), involves cryostat sectioning of a worm “chuck,” followed by Triton X-100 extractions, dounce homogenization, and differential centrifugation. Fractions were collected from each step of the purification process (F1-F14). Notice that fraction F11 was further separated using sucrose gradient fractionation (S1-S14), and results are presented in Fig. 2 (C and D). (C) Portions from fractions F1-F14 were separated on two separate SDS-PAGE gels, transferred to membranes and reacted with either anti-UNC-98 or anti-UNC-97. Note that UNC-98 protein copurifies with the 15,000-g pellet containing the thick filaments (F14; marked with an asterisk). The immunoreactivity in fraction F12 may reflect the possible nuclear localization of UNC-98 described in Mercer et al. (2003). In contrast, UNC-97 protein does not copurify with the 15,000-g pellet containing the thick filaments (F14; marked with an asterisk).