Analysis of lamin B1 phosphorylation on T575.

<p>Human dermal fibroblasts (HDFs) were incubated under the indicated conditions before being fixed and labelled with anti-p-lamin B1 and propidium iodide (PI) to analyse T575 phosphorylation and cell cycle stages respectively. (a) Flow cytometry analysis of cell cycle phases: A representative cell cycle profile based on PI staining is shown. Only G1 cells (left peak with 2N DNA content) were analysed for p-lamin-B1 signals shown in all subsequent analyses. (b) Flow cytometry analysis of G1 interphase Phospho T575 signals: Anti-pT575 signals in the G1-gated population are shown for control cells as well as those treated with MMS, or MMS and kinase inhibitors as indicated. (c) Quantitation of interphase cells with pT575 signal: Summary of percentage of cells above T575 phosphorylation threshold set in (b). Controls without first antibody were used to set the threshold for the pT575 signal gate. There is a significant difference between control and MMS treated cells, and this is also seen in the case of ERK inhibitor (p<0.01) pre-treatment, but no significant difference in the case of the JNK inhibitor pre-treatment (p = 0.06). Data shown is mean +/- s.e.m. (d) Western blotting of G1-gated interphase cells: Sorted G1 cells were lysed and analysed by Western blotting using the anti-p-T575 antibody. The results confirm those observed by flow cytometry. Detection of actin is used to demonstrate equal loading.