Figure 2. Biochemical characterization... | Rockefeller University Press

$Figure 2. Biochemical characterization of the incorporation of the GFP–IC-2C isoform into functional cytoplasmic dynein complexes. (A) Microtubule binding. Western blots of the indicated fractions prepared from PC12 cells with stable expression of GFP–IC-2C were probed with a pan antibody to dynein ICs (74.1) that recognized both the endogenous IC (IC) and GFP-tagged IC (GFP–IC). Taxol-stabilized microtubules were added to the soluble fraction (S), and centrifugation yielded the microtubule depleted supernatant (MDS) and microtubule pellet (MTP). The microtubule pellet was resupended in 10 mM Mg-ATP, and centrifugation yielded the ATP supernatant (ATPS) and microtubule pellet (ATPP). The identity of the GFP–IC-2C bands was confirmed with an antibody to GFP (not depicted). Densitometry was used to estimate that the GFP–IC pool was <15% of the total IC pool. (B) Sucrose density gradient sedimentation. The soluble fraction was also fractionated by sucrose density gradient centrifugation, and the proteins in the fractions were analyzed by SDS-PAGE and Western blotting. The blot was probed with the pan dynein antibody. The fractions at the top (5% sucrose) and bottom (20% sucrose) of the gradient are indicated above the blot. (C) Association with membrane-bounded organelles. PC12 cells were lysed in a cytoplasm-like buffer and fractionated; the postnuclear supernatant (S1) and pellet (P1), high-speed supernatant (S2), and high-speed membrane fraction (P2) are indicated. Immunoprecipitation (IP): magnetic beads, preloaded with antibodies to GFP (αGFP) or control IgG (IgG), were incubated with the membrane fraction, P2. The beads were washed and the fractions were analyzed by SDS-PAGE, and the Western blots were probed with the pan dynein antibody and an antibody to synaptophysin (SY), a membranous organelle marker.$

Figure 2.

Biochemical characterization of the incorporation of the GFP–IC-2C isoform into functional cytoplasmic dynein complexes. (A) Microtubule binding. Western blots of the indicated fractions prepared from PC12 cells with stable expression of GFP–IC-2C were probed with a pan antibody to dynein ICs (74.1) that recognized both the endogenous IC (IC) and GFP-tagged IC (GFP–IC). Taxol-stabilized microtubules were added to the soluble fraction (S), and centrifugation yielded the microtubule depleted supernatant (MDS) and microtubule pellet (MTP). The microtubule pellet was resupended in 10 mM Mg-ATP, and centrifugation yielded the ATP supernatant (ATPS) and microtubule pellet (ATPP). The identity of the GFP–IC-2C bands was confirmed with an antibody to GFP (not depicted). Densitometry was used to estimate that the GFP–IC pool was <15% of the total IC pool. (B) Sucrose density gradient sedimentation. The soluble fraction was also fractionated by sucrose density gradient centrifugation, and the proteins in the fractions were analyzed by SDS-PAGE and Western blotting. The blot was probed with the pan dynein antibody. The fractions at the top (5% sucrose) and bottom (20% sucrose) of the gradient are indicated above the blot. (C) Association with membrane-bounded organelles. PC12 cells were lysed in a cytoplasm-like buffer and fractionated; the postnuclear supernatant (S1) and pellet (P1), high-speed supernatant (S2), and high-speed membrane fraction (P2) are indicated. Immunoprecipitation (IP): magnetic beads, preloaded with antibodies to GFP (αGFP) or control IgG (IgG), were incubated with the membrane fraction, P2. The beads were washed and the fractions were analyzed by SDS-PAGE, and the Western blots were probed with the pan dynein antibody and an antibody to synaptophysin (SY), a membranous organelle marker.

This Feature Is Available To Subscribers Only