Figure 2.

Immunofluorescence of TTLL12, and the effects of TTLL12 silencing and drugs on nitrotyrosine tubulin. (A) Overexpressing cells were used for the immunofluorescence assay to investigate the localization of TTLL12. Scale bar, 25 µm. (B) siTTLL12-1, siTTLL12-2, siLuciferase and siControl cells were treated with 400 µM nitrotyrosine for 24 h. (Ba) Cells from each group were subjected to western blot analysis to determine protein expression. (Bb) The blots were quantified via densitometry and normalized to TBP. Tubulin tyrosine nitration levels of all groups are presented relative to the average tubulin tyrosine nitration level of control siRNAs. Error bars represent the mean ± SEM of three independent experiments. (C) SiTTLL12-1, SiTTLL12-2, SiLuciferase and SiControl cells were treated with 20 µl modified Eagle's medium supplemented with 400 µmol/l nitrotyrosine or 400 µmol/l HCl for 24 h. Cell proliferation was assessed via the MTT assay. The ratio of OD represents the OD 450 nm of treated cells normalized to that of untreated cells. Error bars represent the mean ± SEM of three independent experiments. (D) Stable TTLL12-overexpressing SCC-25 cells were treated with 400 µM nitrotyrosine. Paclitaxel (10 µM) was added to cells in the experimental groups, while nothing was added to the control cells. (a) After 24 h, cells from each group were subjected to western blot analysis to determine protein expression. (b) The blots were quantified via densitometry and normalized to TBP. Nitrotyrosine tubulin levels are presented relative to the average of control. Error bars represent the mean ± SEM of three independent experiments. (E) Cells were treated with 10 µM thiirene. (F) Cells were treated with 10 µM nocodazole. Nitrotyrosine tubulin levels were compared with the treatment and control groups. *P<0.05. TTLL12, tubulin tyrosine ligase like 12; si, small interfering; SEM, standard error of the mean; OD, optical density; TBP, tributyl phosphate; N-tub, nitrotyrosine tubulin.