Detection of p16 expression and the SA-βGal assay reveals p16-dependent senescence. A. Immunohistochemistry was performed as per Lotfi et al, 1997 , using a polyclonal anti-p1 6 antibody (BD Pharmingen, San Diego, CA, USA) 24h post-infection. C6 cells were transduced with the parental pCL virus or with the pCLp16 virus. B. Western blot analysis of 293 cell lysate used as a positive control (lane 1), C6-pCL cell lysate (lane 2) or C6-pCLp16 cell lysate (lane 3). The ECL-biotinylated molecular weight standard (Mw, Amersham Pharmacia Biotech, Upsalla, Sweden) is included for orientation. Lysates from equal numbers of cells were prepared 24h post-infection. The p16 protein was concentrated in an immunoprecipitation step prior to electrophoresis and dectection with a rabbit polyclonal anti-p1 6 antibody (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA). C. The SA-βGal assay  was used to detect senescence associated β-galactosidase activity in C6 cells transduced by either the pCL parental or the pCLp16 virus followed by selection for G418 resistance. Following selection, cells were fixed and stained with x-gal at pH 6.0. At this pH, only senescence associated β-galactosidase activity is detected.