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. 1997 Oct;8(10):2077–2088. doi: 10.1091/mbc.8.10.2077

Figure 4.

Figure 4

Dissociation of cytoplasmic dynein from membranes of cytoplasmic extracts of Xenopus oocytes analyzed by SDS-PAGE immunoblot. (A) Undiluted cytoplasmic extracts of interphase oocytes were supplemented with 0.5 mg/ml monoclonal antibody and incubated on ice for 3 h before resuspending the extract into 58% sucrose acetate buffer. The membrane fractions were isolated by flotation and collected at the 15–50% sucrose interphase. Lane 1, membrane fraction stained with Coomassie; lanes 2–5, immunoblot of membrane fractions stained with dynein IC-specific monoclonal antibody m74–2. Lane 2, membrane fractions of untreated cytoplasmic extract; lane 3, membrane fractions of cytoplasmic extract treated with 0.5 mg/ml m74–2; lane 4, membrane fraction of cytoplasmic extract treated with 0.5 mg/ml m74–1; lane 5, membrane fraction of cytoplasmic extract treated with 0.5 mg/ml control antibody LEP100. Molecular weight markers at left are as follows from top to bottom: 205,000, 97,000, 66,000, and 45,000. (B) Isolated membranes of Xenopus extracts were incubated with antibodies for 3 h on ice and then isolated by centrifugation through a 20% sucrose cushion. Lanes 1 and 1′, membrane fraction treated with 0.5 mg/ml control antibody; lanes 2 and 2′, membrane fraction treated with 0.5 mg/ml m74–2; lanes 1 and 2, stained with dynein IC-specific antibody m74–2; lane 1′ and 2′, stained with monoclonal antibody m150–1 specific for dynactin p150Glued. (C) Immunoblot of antibody-treated membrane fractions from Xenopus extracts stained with polyclonal antibody (polydynH) raised against bacterially expressed dynein heavy chain from Dictyostelium (Vaisberg et al., 1993), dynactin p150Glued-specific monoclonal antibody m150–1, dynein IC-specific monoclonal antibody m74–1, and dynein IC specific monoclonal antibody 70.1 (Steuer et al., 1990). Lane 1, microsomes isolated from bovine brain were used to demonstrate the specificity of the antibodies; lane 2, membrane fraction of cytoplasmic extract from Xenopus oocytes treated with control antibody; lane 3, membrane fraction of cytoplasmic extract of Xenopus oocytes treated with 0.5 mg/ml m74–2.