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. 2009 Jul 14;101(4):722–733. doi: 10.1038/sj.bjc.6605179

Figure 4.

Figure 4

Functional analysis of BRAF and HIPK2 overexpression in hTERT-immortalised astrocytes. (A) Immunofluorescence analysis of hTERT-immortalised astrocytes transfected with CMYC/BRAF or the empty vector (EV) at 48 h using an antibody recognising the C-MYC tag (red), BRAF (green) and DAPI counterstaining (blue). (B) Immunofluorescence analysis of hTERT-immortalised astrocytes transfected with FLAG/HIPK2 or the EV at 48 h using an antibody recognising the FLAG tag (red) and DAPI counterstaining (blue). (C) Proliferation of hTERT-immortalised astrocytes was assessed at 48, 72 and 96 h following transfection with mock, BRAF, HIPK2 or both genes. No difference in the rate of cell growth was observed between the transfectant cells. Results represent the median of three separate experiments performed in triplicates. (C) Total cell extracts of hTERT-immortalised astrocytes transfected with the empty-vector or C-Myc-tagged BRAF. Cells were serum starved overnight before protein lysate extraction. (D) Left panel: hTERT-immortalised astrocytes were transiently transfected with CMYC/BRAF or the empty vector. Cells were serum starved overnight and total protein lysates extracted at 48 h posttransfection. Western blot analysis for C-Myc, BRAF, phopshoERK (pERK) and β-actin (loading control) was performed. Right panel: Empty-vector (EV) and C-Myc/BRAF transfectant hTERT-immortalised astrocytes were stimulated with 200 ng of epidermal growth factor (EGF) for 5 and 15 min. Total cell lysates extracted at baseline (0) and following activation (5, 15 min) with EGF were immunoblotted using antibodies against β-actin (loading control) and phosphorylated ERK (pERK), C-Myc and BRAF. Note the shift of BRAF immunoreactive band following EGF activation which indicates phosphorylation of the kinase and increase in its molecular weight. Importantly, increase in pERK levels relative to baseline following EGF stimulation was two fold higher in C-MYC/BRAF transfectant cells than in EV transfectants. Results represent the median of three separate experiments (P<0.001).