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Fig. 2 Disruption and reformation of the CAV1–ANX2b heterocomplex. (A) Effect of cav1 and anx MO on the formation of the CAV1–ANX2b heterocomplex. Embryos (1–8 cell stage) were injected with the following MO: uninjected (lane 1), cav1 (lane 2), anx2b synthesis 1 (lane 3), anx2b synthesis 2 (lane 4), anx2b mismatched (lane 5), and anx2a (lane 6). 3T3 cell lysate (20 µg) was loaded directly onto the gel as a positive control for ANX2 and CAV1 (lane 7). The embryos were then allowed to develop for 48 h. Larvae were processed to generate lysates (≈20 embryos per sample), and 50 μg of protein was used for IP with CAV1 IgG or ANX2 IgG as indicated. The precipitates were resolved by SDS/PAGE and immunoblotted with ANX2 IgG or CAV IgG as indicated. The data are representative of three to four independent experiments. (B) Reformation of the ANX2b–CAV1 complex in vitro. Embryos (1–8 cell stage) were injected with either cav1 or anx2b MO or uninjected (control) and allowed to develop for 48 h. Lysates were prepared from each class of embryo, and IP were performed as in A. For the last lane, lysates from cav1 MO-injected and anx2b MO-injected embryos were mixed together and incubated at room temperature before IP. SDS/PAGE and immunoblotting are as in A.(C) Rescue of complex formation by anx2b mRNA. Embryos (1–8 cell stage) were injected with anx2b MO ("no RNA" lane) or anx2b MO plus the indicated capped mRNA (control = uninjected). The embryos were allowed to develop for 48 h. Larvae were processed to generate lysates (≈20 embryos per sample), and 50 μg of protein was used for IP with CAV1 IgG (Upper) or ANX2 IgG (Lower). The precipitates were resolved by SDS/PAGE and immunoblotted with the same IgG used for the precipitation. (D) The CAV1–ANX2b complex in embryos is not forming in vitro. Embryos (48 hpf) were collected in a 500-ml Eppendorf tube (20 embryos total per tube), the medium was removed, and the embryos were immediately frozen on dry ice. Specimens were then thawed, lysed, subjected to SDS/PAGE and immunoblotted. For the mix sample, 10 embryos each of anx2b MO-injected and cav1 MO-injected were placed in the tube and frozen as described. No complex formed in the short time that separate ANX2b-free and CAV1-free embryo lysates were mixed together.