Figure 6

TGF-β does not influence glucocorticoid-induced GR α nuclear localization. (A) BEAS-2B cells transiently transfected with a GRα-YFP reporter construct were treated with TGF-β (40 pM) for 24 h then imaged before and after treatment with 30 nM dexamethasone (Dex), using time-lapse live-cell fluorescence microscopy (Leica DMI 6000B). Images were obtained every 30 sec for 30 min prior to Dex treatment, and for 2 h post-treatment. (B) Immunofluorescence of GRα in non-transfected BEAS-2B cells. Cells were incubated using an identical protocol to that in (A) except that at the end of the 2 h dexamethasone incubation, cells were fixed with 10% NBF then GRα immunolocalisation was probed with a FITC-labelled secondary antibody, and nuclei were co-stained with DAPI. Images presented are representative of four independent experiments. (C,D) Protein expression and phosphorylation state of GRα. Cell lysates from BEAS-2B cells were collected after 24 h treatment TGF-β (40pM), then 1 h treatment with 30nM dexamethasone. SDS-PAGE and immunoblotting using phospho-specific antibodies was performed, then the membrane was stripped and re-probed for total GRα expression for normalization. GRα expression was then normalized to β-actin expression. Data are presented as mean and SEM of 4 experiments. *P < 0.05, significantly different from control expression.