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. 2015 Dec 31;291(9):4473–4486. doi: 10.1074/jbc.M115.679035

FIGURE 4.

FIGURE 4.

Overexpressed PrPΔCR and Sho have normal traffic and similar expression levels in SH-SY5Y cells. A, lentiviral transduction to express Shadoo of PrPΔCR cells does not affect its proliferation adversely. Fluorescence of SH/ΔCR, SH/ΔCR+Sho, and SH/ΔCR+PrP cells was measured at every 24th h after attachment for 8 days and in multiple wells, using PrestoBlue reagent. A representative plot is shown. 100% is the fluorescence value at day 0 after attachment for each type of cell. Values are means ± S.D. of replicas in the individual experiment. B, both PrP-WT and PrPΔCR overexpressed in SH-SY5Y cells are complex-glycosylated. PrPΔCR expression levels in SH/ΔCR, SH/ΔCR+mCh, and SH/ΔCR+PrP cells are comparable. Shown is Western blotting analysis of extracts from various cells as indicated above the lanes, untreated (−) or treated with PNGase F (+). Left and right panels are from the same x-ray film, corresponding to the left and right parts of the same membrane. C, Shadoo, overexpressed in SH/ΔCR+Sho cells, is complex-glycosylated. Shown is Western blotting analysis of extracts from SH/ΔCR+mCh and SH/ΔCR+Sho cells, incubated with (+) or without (−) PNGase F or without incubation (0). After PNGase F treatment, the C1 fragment (58) of Shadoo is more readily detectable. D, Shadoo protein is attached to the cell surface via a GPI anchor when expressed in SH-SY5Y cells. Shown is Western blotting analysis of cell lysates (Cl) and supernatant medium (M) from SH/GFP (left) and SH/Sho (right) cells, incubated with (+) or without (−) PI-PLC. Endogenous Shadoo expression is below the detection limit. The amount of Shadoo decreases below the detection limit in the cell samples of PI-PLC-treated cells (C− versus C+), whereas the amount of an intracellular protein, β-actin, remains unchanged. Shadoo remains below the detection limit in the medium samples. Left and right panels are from the same x-ray film, corresponding to the left and right parts of the same membrane. E, positive control of PI-PLC treatment for D; PrP-WT is attached to the cell surface via a GPI anchor. Shown is Western blotting analysis of cell lysate (Cl) and supernatant medium (M) from SH/PrP cells, untreated (−) or treated (+) with PI-PLC. A decrease in the PrP level is apparent in the lysates. Unlike Shadoo, PrP can be detected in the medium after PI-PLC treatment. F, PrPΔCR expression level in SH/ΔCR+PrP is similar to PrP-WT and is lower than in SH/ΔCR+Sho cells. Comparison was carried out by Western blotting analysis of serial dilutions of extracts from SH/ΔCR+PrP (12, 6, and 3 μg of total protein; left) and of extracts from SH/ΔCR+Sho (24, 12, 6, and 3 μg of total protein; right) treated with PNGase F. Left and right panels are from the same x-ray film, corresponding to the left and right parts of the same membrane. G, schematic design of rSho-PrP recombinant polypeptide. The mSho(81–116) fragment is inserted into mPrP(23–231) between the 93rd and 94th amino acids in order to produce a bacterially expressed polypeptide that contains epitopes for both Sho and PrP antibodies to compare relative Shadoo and PrP protein levels. H, PrPΔCR and Shadoo expression levels of SH/ΔCR+Sho cells are similar. Comparison was carried out by Western blotting analysis of serial dilutions of extracts from SH/ΔCR+Sho cells, treated with PNGase F, (24, 12, 6, or 3 μg of total protein; left) and of rSho-PrP recombinant polypeptide (200, 100, and 50 ng of purified recombinant protein; right). Left and right panels are from the same x-ray film, corresponding to the left and right parts of the same membrane. B–F and H, *, a nonspecific band. Numbers and marks on the left indicate the positions of the corresponding molecular size markers in kDa. As a loading control, β-actin was used.