Fig. 6.

Silencing of PDZD11 with small interfering (si)RNAs decreases carrier-mediated biotin uptake in HuTu-80 cells. HuTu-80 cells were treated with the PDZD11-specific siRNAs or with the nontargeting negative control siRNA (control) as described in experimental procedures. A: initial rate of carrier-mediated biotin uptake by HuTu-80 cells. Cells were incubated at 37^°C in Krebs-Ringer buffer (pH 7.4). [³H]biotin (9 nM) was added to the incubation medium with or without unlabeled biotin at the onset of incubation. Uptake was measured after 4 min of incubation. Carrier-mediated component was calculated as described in experimental procedures. Data are means ± SE of at least 3 separate uptake determinations. B: Western blot analysis was performed using 30 μg protein from the cell lysates. Blot was probed with the polyclonal antibodies directed against PDZD11 or hSMVT, and data were normalized to the amount of β-actin. Data presented are from the representative set of experiments. C: total RNA was isolated and real-time PCR was performed using gene-specific primers. Data are from 3 different experiments and expressed relative to 18S rRNA as means ± SE. Note that some statistical SEs of the mean bars are not visible on the scale of the graph presented. *P < 0.01.