pFN is a substrate for FXIII-A activity. (A) FN detection in total cell protein extracts by enzyme-linked immunosorbent assay during differentiation of preadipocytes to adipocytes during 8 days. FN levels increase in preadipocyte layers during early differentiation and peak at day 2. (B) Affinity-purified preadipocyte culture extracts labeled with BPA shows its incorporation into total FN but not into cFN (EDA-FN), thus demonstrating that cFN/EDA-FN is not a TG substrate and suggesting that pFN is the main crosslinking target in preadipocytes. Total cell extract (Tot.CE) was used as positive control. (C) The FXIII-A–specific substrate peptide bF11 was able to pull down FN, demonstrating that it acts as a specific FXIII-A substrate in preadipocytes. NC9 blocks bF11-mediated FN labeling. The control peptide bF11QN shows no labeling. (D) Immunofluorescence microscopy of bpFN (green) in preadipocytes (actin, red) treated with basic cell culture media (M) (serum-free conditions). Inhibition of TG activity by NC9 decreased bpFN matrix levels (green) in preadipocytes. Nuclei are stained with DAPI (blue). (E) Analysis of exogenous bpFN levels in media using enzyme-linked immunosorbent assay after 24-hour incubation with preadipocytes during differentiation shows a significant increase in media upon NC9 treatment at day 1, indicating that less pFN is incorporating as extracellular matrix. (F) Quantification of intracellular FN levels analyzed from trypsinized cells shows no change in FN levels in cells upon NC9 treatment. (G-H) Assembly of pFN into preadipocyte extracellular matrix is impaired by NC9 treatment. Exogenous bpFN was given to the cells for 24 hours followed by its detection prepared with DOC (DOC-sol) and sodium dodecyl sulfate-containing (DOC-insol) buffers. Quantification was done after WB and detection of biotin. icFN, intracellular FN. All error bars represent SEM (n = 3). *P < .05; **P < .01; ***P < .001.