Figure 2

Localisation of TIMP-3 in IPAH lung tissue. Immunocytochemistry for TIMP-3 was carried out using an polyclonal antisera raised against the human carboxy-terminal peptide of TIMP-3. Plexiform lesion showing location of TIMP-3 in subendothelium. (A). Hypertrophied artery, showing location of TIMP-3 in in vascular smooth muscle cells and myoblasts (B). Scale bars = 100 μm. Protein extracted from IPAH and donor lung tissues was analysed by western blot using an antiserum to TIMP-3 (C). The antibody recognises both the unglycosalated form (24 kDa), and the glycosalated form of TIMP-3 (30 kDa). Expression of TIMP-3 in human cells (D). RNA extracted from human cell lines and explant derived cells was reverse transcribed and amplified by PCR and run on agarose/ethidium bromide stained gels. RT-PCR for TIMP-3 (top panel) and G3PDH (bottom panel). The cell types examined were: Lanes; 1, HL60 (promyelocytic leukaemia); 2, Daudi (Burkitt's lymphoma); 3, EB-transformed lymphocytes; 4, K562 (erythroleukemia) 5, pulmonary adult fibroblasts; 6, pulmonary artery smooth muscle; 7, bronchial smooth muscle; 8, A549 (adenocarcinoma alveolar epithelial); 9, H322 (adenocarcinoma bronchial epithelial); 10, placental microvascular endothelial; 11, umbilical vein endothelial; -, no cDNA negative control; + control lung cDNA; M, phiX174 DNA/HaeIII markers.