Fig 1: 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.
Fig 2: Effect of overexpressing TTLL12 and nitrotyrosine on the proliferation of SCC-25 cells. (A) Cells were subjected to western blot analysis to determine protein expression. (B) Cell proliferation was assessed via the MTT assay every 24 h. The ratio of OD 450 nm was calculated as OD assay/OD control. Error bars represent the mean ± SEM of three independent experiments. (C) TTLL12-overexpressing clones, TTLL12_A and TTLL12_B, as well as control_A and control_B cells were treated with 400 µM nitrotyrosine for 24 h. (Ca) Cells from each group were subjected to western blot analysis to determine protein expression. (Cb) 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_A. Error bars represent the mean ± SEM of three independent experiments. (D) TTLL12_A and control_A cells were cultured in different concentrations of nitrotyrosine (0–400 µM) for 24 h. Scale bar, 100 µm. (E) TTLL12_A, TTLL12_B, control_A and control_B cells were treated with 20 µl modified Eagle's medium supplemented with 400 µmol/l nitrotyrosine for 24 h, while the untreated group received medium supplemented with HCl. Cells proliferation was assessed via the MTT assay. The ratio of OD 450 nm represents the OD 450 nm of treated cells normalized to that of untreated cells. Error bars are represent the mean ± SEM of three independent experiments. *P<0.05. TTLL12, tubulin tyrosine ligase like 12; OD, optical density; TBP, tributyl phosphate, SEM, standard error of the mean; N-tub, nitrotyrosine tubulin.
Supplier Page from Abcam for Anti-TATA binding protein TBP antibody [EPR21954] - ChIP Grade