The anti-V5 antibody was also used to detect V5-tagged protein by Western blot. Cell lysates were prepared following transfection using lysis buffer (0.15 M sodium chloride, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS, 50 mM Tris-Cl pH 8.0) and protease inhibitor cocktail (Sigma). Lysates were incubated for 15 min on ice followed by sonication to shear genomic DNA. Following Bradford analysis equal protein content from JV V5 and JV FLC lysates were run on a 10% SDS-PAGE gel and subsequently transferred onto Immobilon-P PVDF membrane (Millipore) according to the manufacturer's recommendations.