Biochemical and Biophysical Research Communications
Mammalian actin binding protein 1 is essential for endocytosis but not lamellipodia formation: functional analysis by RNA interference☆
Section snippets
Materials and methods
Antibodies and other reagents. The polyclonal anti-mAbp1antibody was produced by immunizing rabbit with a glutathione S-transferase (GST) fusion protein encompassing amino acids 146–326 of human mAbp1. For immunofluorescence, the antibody was purified using CNBr-activated Sepharose immobilized with the immunogen. Anti-Erk and anti-CrkL (Santa Cruz Biotechnology, CA) were rabbit polyclonal antibodies and anti-Akt (Santa Cruz Biotechnology) was goat polyclonal. Mouse monoclonal antibodies
Results and discussion
To knock down mAbp1 protein expression, a synthetic double-stranded siRNA directed to the gene sequence of human mAbp1 was transfected into 293T cells together with EGFP-expression plasmid utilized to monitor cells receiving the siRNA. As a negative control, cells were transfected with siRNA to the Dok2 gene [16], which is not expressed in 293T cells (data not shown). Forty-eight hours after transfection, mAbp1 was visualized by immunofluorescence using an affinity-purified anti-mAbp1
Acknowledgements
We thank Drs. F. Michel and T. Tuschl for help with some experiments and suggestions. We also thank Drs. G. Langsley, S. Pellegrini, and F. Michel for critical reading of the manuscript. S.M.-O. was a recipient of an Association pour la Recherche sur le Cancer fellowship. This work was supported by grants from the Institut Pasteur and the Centre National pour la Recherche Scientifique.
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Abbreviations: mAbp1, mammalian actin binding protein 1; siRNA, short interfering RNA; Tf, transferrin; RNAi, RNA interference, EGFP, enhanced green fluorescent protein.