Collagen processing determines the sheet forming capacity in AF clones.
A) Immunoblot analyses of Collagen type I protein in AF-S and AF-nS clones cultured in control or TGFβ3 containing medium for 7 days. Procollagen-alpha 1 (180 kDa; pro-α1) and Procollagen alpha 2 (145 kDa; pro-α2) variants of Collagen type I are indicated. The appearance of mature alpha 1 (135 kDa; α1) and alpha 2 (120 kDa; α2) variants of Collagen type I correlated well with sheet formation in AF-S clones. β-Actin (βACT) was used as loading control. B) Immunoblot analyses of Collagen type I protein in AF-S clones cultured in TGFβ3 medium with or without ascorbic acid for 7 days. Alpha-tubulin (αTUB) was used as loading control. C) Quantification of Collagen maturation as a function of time in AF-S and AF-nS clones. The ratio of mature COL1A1 over ProCOL1A1 and mature COL1A2 over ProCOL1A2 are depicted in the left and right graphs, respectively. At t = 0 no mature forms of Collagen type I were detectable. Statistical significance was assessed by Student’s t-test; * p<0.05.