Fig. 2. (A) Hydrodynamic properties of dynein 1 and dynein 2 subunits. Rat testis cytosolic extract was centrifuged on a 5-20% sucrose gradient and analyzed by western blotting. On the left panel, total extract was probed with antibodies against dynein 2 HC (D2), LIC3 (L3), dynein 1 HC (D1), dynein 1 IC (IC), dynein 1 LIC 1 and 2 (L1) and the mammalian orthologue CGI-53 (C5) of the p52 subunit of the Chlamydomonas IFT particle (Cole et al., 1998). Except for the anti-LIC1 Ab, which detects both LIC1 and LIC2 (Tynan et al., 2000a), a single band was detected with each antibody; distinct mobilities detected for dynein 1 HC and dynein 2 HC on 5% SDS-PAGE (inset). Observed M_r on SDS-PAGE using molecular mass standards was 48x10³ for CGI-53 and 40x10³ for LIC3, close to their predicted M_r of 50x10³ and 40x10³, respectively. On the right panel, sucrose gradient pellet (Plt), and even-numbered fractions of the same extract were probed. D2 HC and LIC3 co-migrated as a peak between fractions 6 and 8 (calculated S value: 17S). D1 HC, IC 70, LIC 1 and 2 co-migrated as a peak between fractions 2 to 4 (22S). The CGI-53 IFT particle component peaked at 19S. The intensity of the bands for each polypeptide species were quantified and plotted below in arbitrary units. (B) Co-immunoprecipitation of dynein 2 components. Rat testis extract was immunoprecipitated with either anti-dynein 2 HC Ab (D2 IP), anti-LIC3 Ab (LIC3 IP), or without Ab (beads only), and immunoblotted using anti-dynein 2 HC Ab (left) or anti-LIC3 (right). Anti-dynein 2 HC Ab co-immunoprecipitated LIC3 (1st lane on the right panel), and anti-LIC3 Ab co-immunoprecipitated dynein 2 HC albeit at lower levels (2nd lane on the left panel).