Fig. 8. The deposition of laminin 5 is regulated independently of secretion. (A) ELISA assay for laminin 5 deposits confirmed loss of deposition. Cells were culture for 4 hours on laminin 5 then removed with 5 mM EDTA in PBS for 30 minutes at 37°C and new laminin 5 deposits were quantified by ELISA. Signals were normalized relative to D2-1, DMSO=1.0. No cells (–), DMSO treated cells (nt), 1 µM nocodazole (noc), 1 µM taxol (tax) or 6 µM rottlerin (rot). Presented are the average and standard deviation of three wells from a representative experiment. (B) Secretion of laminin 5 into the culture medium was not blocked by nocodazole. Keratinocytes labeled with [³⁵S]methionine were adhered to exogenous laminin 5 for 3 hours in the presence of 0, 0.1, 0.5 or 1.0 µM nocodazole (lanes 1-4 and lanes 5-8) and the conditioned media were collected. Immunoprecipitation of secreted laminin 5 from cell culture media was performed in RIPA buffer using mAb C2-5 against the laminin 3 chain (lanes 1-4) or mAb B4-6 against the laminin 2 chain (lanes 5-8). Molecular masses (in kDa) of the three chains of the laminin 5 trimer are indicated.