Ctgf blocking antibody

CTGF levels in media from fibroblasts treated with keratinocyte-CM were determined by measuring the levels of CTGF in media using specific ELISA kit (PeproTech). Collagen levels in fibroblasts were measured as follows. Fibroblasts were first starved in 0.1% FBS for 24 hours, then treated with keratinocyte-CM with/without TGFβ1 (R&D Systems) or/and CTGF blocking antibody (Santa Cruz) for 72 hours. Supernatant was collected to measure collagen by ELISA (Biomatik).

The effect of keratinocyte-CM on the proliferation of fibroblasts and MSCs were determined with a BrdU Cell Proliferation Assay Kit (Cell Signaling Technology). CM were then collected from keratinocytes transfected with FOXO1 siRNA or scrambled control siRNA, and incubated with fibroblasts or MSCs in 96-well plates with/without TGFβ1 antibody (R&D Systems) or/and CTGF blocking antibody (Santa Cruz) for 72 hours, and incubated with 10 μM BrdU for 6 hours. Cells were fixed with cold methanol and incubated with anti-BrdU antibody. BrdU incorporation was determined by measuring absorbance at 450 nm. Experiments were repeated at least three times.