EDEM1 knockdown results in the accumulation of P23H rod opsin. P23H rod
opsin-GFP (P23H-GFP) and empty vector (A) or P23H-GFP and shEDEM1 (B) were
transfected in SK-N-SH cells. 24 hours after transfection, the cells were
fixed and analysed by confocal microscopy. Scanning parameters during image
acquisition were kept constant. Arrows indicate increased ER staining and
inclusion formation. Scale bar: 10 μm. (C) The incidence of inclusion
formation of P23H-GFP was assessed with increasing shEDEM1 plasmid (1:1, 1:2,
1:3, 1:4). Error bars represent ± 2 s.e.m.
*P=0.0134. (D) Constant pMT3-P23H rod opsin and increasing
(1:2, 1:3, 1:4) molar plasmid equivalents of shEDEM1 were transfected in
SK-N-SH cells. DM-soluble fractions were obtained 24 hours after transfection
and 10 μg protein resolved by SDS-PAGE. Asterisk indicates species showing
greatest accumulation. Rod opsin was detected using 1D4 mAb. The position of
molecular size markers in kDa are indicated on the left.