Fig. 6.

CCN5 shRNA knockdown does not alter podosome formation. a VSMCs were infected with either lentivirus expressing shRNA against CCN5 (lane 1) or lentivirus expressing GFP (lane 2). Cell lysates were then prepared from a fraction of the infected cells in an NP-40 based lysis buffer. Proteins were resolved by SDS-PAGE and analyzed by Western blotting using an antibody against CCN5 or GAPDH (loading control) b The level of CCN5 from a was quantitated using densitometry. c. Another fraction of the cells infected with lentivirus (both CCN5 shRNA and GFP), as well as a group of uninfected cells were trypsinized and plated on glass slides. Half of each of the aforementioned three groups of cells were then treated with 2 μM phorbol ester phorbol 12,13-dibutyrate (PDBU) for 1 h to induce podosome formation, while the other half was untreated Cells were then fixed and stained for cortactin (podosomal marker). Percentage of cells containing podosomes were then calculated and graphed. Error Bars = standard deviation, n = 3