Figure 4. d- cer-C6 treatment affects... | Rockefeller University Press

Figure 4.

$Figure 4. d-cer-C6 treatment affects TGN46 glycosylation. (A) HeLa cells were treated with ethanol, the indicated concentrations of d-cer-C6, or 20 µM l-cer-C6 for 4 h, after which the cells were lysed, and the lysates were analyzed by Western blotting using an anti-TGN46 antibody. A 110-kD band corresponds to the fully processed, fully glycosylated TGN46, whereas smaller bands of ∼95, 80, and 75 kD correspond to immature forms of TGN46. (B) HeLa cells were treated with ethanol or 20 µM d-cer-C6 for 4 h, after which the cells were biotinylated. After isolation of biotinylated proteins, the biotinylated fractions and cell lysates were treated with neuraminidase (Neur.) or buffer alone and analyzed by Western blotting using antibodies against TGN46 and β-actin. (C) HeLa cells were treated with ethanol or with 20 µM d-cer-C6 for 4 h, after which the cells were lysed. Lysates were treated with a deglycosylation (Deglyc.) mix (second and fifth lanes), treated with neuraminidase (third and sixth lanes), or remained untreated (first and fourth lanes) and analyzed by Western blotting using an anti-TGN46 antibody. (D) HeLa cells transfected with control (CTRL) or SMS1 + SMS2 siRNA for 92 h were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h, after which cells were lysed, and the lysates were analyzed by Western blotting using anti-TGN46 and anti–β-actin antibodies. (E) Quantitation of the band intensity of mature 110-kD TGN46 (in percentages of total TGN46) for the experiment in D. Bars show the mean values ± SEM of four independent experiments (n = 4). Statistical significance is indicated as **, P < 0.01 or n.s., P > 0.05.$

d-cer-C6 treatment affects TGN46 glycosylation. (A) HeLa cells were treated with ethanol, the indicated concentrations of d-cer-C6, or 20 µM l-cer-C6 for 4 h, after which the cells were lysed, and the lysates were analyzed by Western blotting using an anti-TGN46 antibody. A 110-kD band corresponds to the fully processed, fully glycosylated TGN46, whereas smaller bands of ∼95, 80, and 75 kD correspond to immature forms of TGN46. (B) HeLa cells were treated with ethanol or 20 µM d-cer-C6 for 4 h, after which the cells were biotinylated. After isolation of biotinylated proteins, the biotinylated fractions and cell lysates were treated with neuraminidase (Neur.) or buffer alone and analyzed by Western blotting using antibodies against TGN46 and β-actin. (C) HeLa cells were treated with ethanol or with 20 µM d-cer-C6 for 4 h, after which the cells were lysed. Lysates were treated with a deglycosylation (Deglyc.) mix (second and fifth lanes), treated with neuraminidase (third and sixth lanes), or remained untreated (first and fourth lanes) and analyzed by Western blotting using an anti-TGN46 antibody. (D) HeLa cells transfected with control (CTRL) or SMS1 + SMS2 siRNA for 92 h were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h, after which cells were lysed, and the lysates were analyzed by Western blotting using anti-TGN46 and anti–β-actin antibodies. (E) Quantitation of the band intensity of mature 110-kD TGN46 (in percentages of total TGN46) for the experiment in D. Bars show the mean values ± SEM of four independent experiments (n = 4). Statistical significance is indicated as **, P < 0.01 or n.s., P > 0.05.

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