Figure 6. Effects of inhibitors on levels of expressed myocilin-GFP in inducible cells.

Fluorescence images of myocilin_WT-GFP (Myoc_WT-GFP, A), myocilin_P370L-GFP (Myoc_P370L-GFP, C), myocilin_Q368X-GFP (Myoc_Q368X-GFP, E)-expressing-RGC5 cells treated with various inhibitors are shown. Scale bar, 20 µm. For Western blotting, RGC5 cells induced by Dox for 24 h to express myocilin_WT-GFP (B), myocilin_P370L-GFP (D), and myocilin_Q368X-GFP (F) were treated for 16 h with vehicle DMSO, or proteasomal (LCT and epoxomicin), lysosomal (NH₄Cl and chloroquin) or autophagic (3-MA) inhibitors. Proteins (25 µg) in cell lysates were immunoblotted with anti-myocilin or anti-GAPDH. Protein bands for the endogenous myocilin (55/57 kDa) and myocilin-GFP (∼100 kDa) were seen. Densitometry was performed. The myocilin-GFP/GAPDH relative to the DMSO control ratios are presented. All experiments were repeated at least 3 times.