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Fig. 3. Knockdown of TTC3 in differentiated PC12 cells. (A) HEK293 cells were co-transfected with GFP-TTC3 and unrelated pDECAP control (pD) or pDECAP-TTC3 (pDT) plasmids. The expression of GFP-TTC3 and that of an internal loading control (Vinculin) were then evaluated by western blot 48 hours after transfection. (B) HEK293 cells were co-transfected with GFP-TTC3 and pCMV-GIN-ZEO sh-mir plasmids, expressing a double mismatch (pC) and two perfect match (pC-1 and pC-2) interfering RNAs. The reduction of GFP-TTC3 was evaluated as in A. (C) PC12 cells were transfected with the indicated control and RNAi plasmids. The levels of endogenous TTC3 mRNA were evaluated 48 hours after transfection by semi-quantitative RT-PCR, using internal control 
-actin primers. (D,E) PC12 cells were transfected with the indicated control and RNAi plasmids and treated with NGF for 3 days. Cells were then fixed, stained with phalloidin and analyzed by IF. The ratio between length of the main neurite and principal diameter of cell body was determined for at least 200 cells. Histograms represent the average of three independent experiments (D). The differences between control (gray bars) and RNAi (black bars) samples were statistically significant, as determined by the Student's t-test (P<0.0001). Error bars represent s.d. Bar, 10 µm.
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