Figure 4.

Cellular localization of overexpressed OPTN. A: TM cells were transfected with pEGFP-N1 or pOPTN_WT-GFP for 16 hours. a: Twenty μg of protein extracts from pEGFP-N1-transfected (lane 1) and pOPTN_WT-GFP-transfected (lane 2) cells were subjected to Western blotting with anti-OPTN (α-OPTN), anti-GFP (α-GFP), or anti-GAPDH (α-GAPDH). b–e: TM cells were transfected with pOPTN_WT-GFP, fixed, and immunostained with anti-golgin97 (c; red). OPTN foci (green) are seen in b, d, and e. The Golgi apparatus is presented at a higher magnification in the inset in c. Merged image is shown in d. The boxed perinuclear/Golgi region in d is enlarged in e. OPTN foci were predominantly distributed in the perinuclear region around the Golgi with little colocalization observed with the Golgi marker golgin97. Scale bars: 10 μm (b–d); 4 μm (e). B: RPE cells were transfected with 0.4 μg (a) or 2 μg (b) of pOPTN_WT-GFP or pEGFP-N1 per milliliter of transfection solution. a: Twenty μg of protein extracts from pEGFP-N1- (lane 1) and pOPTN_WT-GFP-transfected cells (lane 2) were immunoblotted with α-OPTN, α-GFP, or α-GAPDH. b: Ten μg (1/20 of total lysate) of protein extracts (lanes 1 and 3) and one-sixth of concentrated media (lanes 2 and 4) from pEGFP-N1-transfected (lanes 1 and 2) and pOPTN_WT-GFP-transfected (lanes 3 and 4) cultures were probed with α-OPTN and α-GFP. In parallel experiments, the pOPTN_WT-GFP-transfected (c–e; GFP in green) and pEGFP-N1-transfected (f–h; GFP in green) cells were immunostained with anti-golgin97 (d and g; red). The Golgi apparatus is shown at a higher magnification in insets in d and g. Merged images are presented in e and h. Images were taken using a 63× oil objective. Bar = 10 μm.