Fig. 5.

Abi1 knockdown in MDA-MB-231 cells inhibited Src activation, Id1 expression, cell migration and proliferation. (A) Knockdown of Abi1 expression inhibited Src activation and Id1 expression. Left panel shows MDA-MB-231 control and Abi1KD cells were lysed and immunoprecipitated with anti-Src antibody. The immunoprecipitates were separated on SDS–polyacrylamide gel electrophoresis (PAGE) and subjected to western blot analysis using anti-Src p416 antibody that recognize active Src, as indicated by an arrowhead. The membrane was then stripped and reprobed with the antibody that recognizes total Src protein, as indicated by an arrow. Right panel shows control and Abi1KD cells were lysed, separated on SDS–PAGE and subjected to western blot analysis using indicated antibodies. (B) Abi1 knockdown inhibited MDA-MB-231 cell migration. A total of 5 × 10⁴ MDA-MB-231 control and Abi1KD cells were tested for their migratory abilities using the Boyden chamber migration assay. The y-axis represents the percentage of the cells that migrated to the other side of insert membranes and is the mean ± SD of triplicate inserts. The upper panel shows a representative field of the insert membranes with migrated cells; *P < 0.01. (C) Abi1 gene silencing reduced cell proliferation in MDA-MB-231 cells. A total of 1 × 10⁴ control and Abk1KD cells were grown in six-well plate for indicated time periods. Cells were harvested at the end of the time period and cell numbers in each well were counted and expressed as mean ± SD of triplicate wells.