Fig. 5. (A) Effect of PD153035 and TZ on the localisation of PPAR. NHU cells were seeded at 2x10⁵ cells/ml onto glass slides, allowed to attach and treated for 4 hours with or without PD153035 (1 µM) in the presence or absence of TZ (1 µM). The slides were fixed and immunofluorescence was performed for PPAR, with nuclei counterstained using Hoechst 33258. Bar, 100 µm. Western blot analysis was used to show the effect of EGFR inhibition on the phosphorylation of ERK (B) or PPAR (C). NHU cells were treated with (+) or without (–) PD153035 (1 µM) for 4 hours. (B) Protein lysate (40 µg) from each sample was used to analyse phospho- and total ERK, as described in Materials and Methods. The data are representative of three separate experiments. (C) Protein lysate (200 µg) was used to immunoprecipitate with PPAR-agarose conjugate (10 µg), as outlined in the Materials and Methods, before being resolved on an 10% SDS-PAGE and transferred to nitrocellulose; phospho-serine or PPAR was detected using specific antibodies and enhanced chemiluminescence.