Figure 2.

Expression of transforming growth factor β (TGFβ) signalling components in neuroendocrine tumour (NET) cell lines. (A) NET cell lines express TGFβ type I receptor (TGFβR I) and TGFβ type II receptor (TGFβR II) mRNA transcripts. RT-PCR analysis using primers specifically directed against TGFβR I, TGFβR II, and GAPDH was performed. Alternating lanes represent the results with (+) or without (−) prior reverse transcription to exclude genomic DNA contamination. A 100 bp DNA standard was used for size determination and the size of the amplicons are indicated. (B) Immunoblots using whole cell extracts (50 μg protein/lane) were performed to determine expression of TGFβR I, TGFβR II, Smad2, Smad3, and Smad4 protein. Immunodetectable proteins migrated at the expected molecular mass, as indicated. (C) TGFβ-1 protein is expressed and secreted in NET cell lines. Cells (2×10⁴) were seeded overnight and cultured in serum free UltraCulture medium for three days. A TGFβ-1 specific ELISA was used to determine TGFβ-1 concentrations in the supernatants of the indicated cell lines and normalised to protein content/ml supernatant. Each experiment was performed in triplicate. (D) NET cell lines express TGFβ-2 and TGFβ-3 mRNA transcripts. RT-PCR analysis using primers specifically directed against TGFβ-2 and TGFβ-3 was performed. Alternating lanes represent the results with (+) or without (−) prior reverse transcription.