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Fig. 7. Expression of myc-tagged CRB3 mutants in MCF10A cells. (A) Amino acid sequences of the CRB3 mutants. The intracellular amino acid sequences of the myc-tagged CRB3 mutants are shown. The putative FERM binding motif is underlined, conserved RxPPxP motif is in bold, and alanine substitutions are highlighted in red. (B) Retroviral-mediated expression of CRB3 mutants in MCF10A cells. Retroviruses were constructed as described in Materials and Methods, and MCF10A cells were infected as before. Stably expressing pools were isolated. Lysates were prepared from confluent plates. 30 µg protein lysate from each cell line were separated by Nu-PAGE electrophoresis and analyzed by western blotting for the myc epitope using the 4A6 anti-myc antibody. (C) Mutations in the FERM binding motif and RxPPxP motif do not affect binding of mycCRB3 to PALS1, but deletion of the ERLI sequence does. Lysates were prepared from confluent plates of the indicated cell lines, and 1 mg of each lysate sample was immunoprecipitated overnight with the 9E10 anti-myc antibody. Immunoprecipitates were washed, resolved by NuPAGE gel electrophoresis, transferred to nitrocellulose membranes, and blotted for PALS1. PALS1 runs as a characteristic doublet. The positions of molecular weight markers in kDa are indicated on the left.
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