Figure 5.

Myocilin was secreted continuously in pulse chase experiments. MDCK cells in 6-well plates were labeled with ³⁵S cysteine/methionine. Following a 30 min pulse, cells were washed and then chased with 1.5 ml DMEM containing a 10 fold excess of cysteine-methionine and 10% FBS for 0, 0.5, 1, 2, 6, 9, 15, 18, 24, and 48 h. Myocilin protein was immunoprecipitated from cell lysates (upper panels) or medium (lower panels). Samples were separated by 4-20% SDS-PAGE. Myocilin closely migrated as doublet bands with excess nonradioactive sheep immunoglobulin heavy chain, which influenced the resolution of the myocilin bands in some lanes. Myocilin immunoprecipitated from chase medium was detectable after 30 min and continued to be secreted for 48 h.