Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - (SuppFig1.pdf; 29 KB) Recombinant human CTGF (rhCTGF) was purified by Heparin Sepharose affinity chromatography followed by Q-CM tandem column ion exchange chromatography and resolved by SDS-PAGE (4-12% gradient Bis-Tris gel) under non-reducing conditions. Staining by silver and Coomassie blue was conducted by standard methods. In both the silver and Coomassie stained gels, three distinct bands can be seen, two having molecular weights of 31-36.5 kDa and one with molecular weight ~14.4 kDa. Based on binding to a ConA Sepharose column, the uppermost band at ~ 36.5 kDa is glycosylated full length rhCTGF, and the lower molecular weight band, which did not bind ConA Sepharose, is non-glycosylated full length rhCTGF. rhCTGF was resolved on a non-reducing gel as described above, electroblotted onto nitrocellulose and probed for immunoreactive species using antibodies reactive with the N-terminal (FibroGen #19) and the C-terminal halves of CTGF (FibroGen #839). Detection and visualization was by HRP-conjugated goat anti-human or anti-rabbit antibody. Both CTGF antibodies recognized the two bands at 31-36.5 kDa. Only the C-terminal reactive antibody recognized the ~14.4 kDa band. The molecular weight and reactivity with the C-terminal antibody is consistent with the identification of this band as the C-terminal half of CTGF.
• Supplemental Figure 4B i - (SuppFig4Bi.pdf; 600 KB)
• Supplemental Figure 4B i - (SuppFig4Bii.pdf; 1.23 MB)
• Supplemental Movie 1 - ( SuppMovie1.mov; 52.5 MB)
• Supplemental Movie 2 - ( SuppMovie2.avi; 123 MB)
  Legend for Supplemental Figures 4 i, 4 ii & Supplemental Movies 1, 2: CTGF stimulated a rapid disassembly of the actin cytoskeleton accompanied by the adoption of a polarised morphology with clear accumulation of actin at the leading edge in cells transfected with GFP-actin (4 i, and Supplemental Movie 1). In contrast, the actin cytoskeleton remained intact and there was no polarised morphology in cells co-transfected with GFP-actin and either mutant; expression of the YFP- p27^Kipl mutants was again confined to the nucleus. (4 ii, and Supplemental Movie 2).