Fig. 2
Overexpression of TTLL4 in MDA-MB231 cells increases MT-glutamylation. a TTLL4 was overexpressed by using a lentiviral vector. Success of overexpression was analyzed by real-time PCR. Shown are mean values ± SD of three different experiments. Wt = untreated control cells, control = cells treated with Lego vector, TTLL4 = cells treated with Lego vector encoding for TTLL4. b Polyglutamylated proteins were immunoprecipitated from control and TTLL4plus cells, using an antibody against polyglutamylation modification (GT335). Cell lysates (“input”), supernatants (“S/N”) from cell lysates incubated with GT335-coupled beads and proteins bound to GT335 coupled beads (“beads”) were analyzed by Western blotting for ß-tubulin and NAP-1 levels. IgG signals served as loading control. The lower band appearing in the NAP-1 blot is a residue signal of β-tubulin antibody because the same membrane was used to probe against all 3 proteins. c Fixed cells were labeled using the GT335 antibody (green), ß-tubulin antibody (red) and DAPI (blue) to mark nuclei. Bar: 20 μm. Fluorescence was analyzed by confocal microscopy. Right panel: Fluorescence of GT335 and ß-tubulin signals were analyzed and the ratio GT335/ß-tubulin (Glutamyated MTs) was calculated. Shown are mean values ± SD of 40 cells