Figure 6

Analysis of profilin-1 expression in T-cell clones obtained from octogenarian and centenarian donors. For Western blot analysis, one million cells from T-cell clones obtained from octogenarian and centenarian donors at early, middle and late passage were pelleted and lysed in sample buffer (50 mM Tris, pH 8.0, 120 mM NaCl, 0.5% NP40, 10 μg/mL PMSF (phenylmethylsulfonylfluoride), and 1× protease inhibitor cocktail. A total of 30 μg protein was suspended in laemmli buffer (50 mM tris, pH 6.8, 1% B-mercaptoethanol, 2% sodium dodecyl sulphate, 0.1% bromophenol blue, and 10% glycerol). Samples were boiled and subject to Western blot analysis on NuPAGE™ Bis-Tris gels with MES running buffer. Human profilin-1 and β-actin proteins were detected and visualised by enhanced chemi-luminescence. Protein expression profiles from one representative clone from an octogenarian and centenarian donor is shown along with population doublings (PD) for each sample analysed (A). Average profilin-1 expression in early and late passage octogenarian and centenarian samples is depicted graphically following normalisation to β-actin expression in each sample (B). Student's t-test was performed and p < 0.05 was considered statistically significant.