Anti detyrosinated tubulin

The following primary antibodies were used: mouse monoclonal anti-E-Cadherin (BD Biosciences, Cat. No. 610181, 1:200), rabbit polyclonal anti-Ki 67 (Cell Signaling, Cat. No. 9027, 1:100), rabbit polyclonal anti-β-Catenin (Sigma, Cat. No. C2206, 1:50), rabbit polyclonal anti-detyrosinated tubulin (Abcam, Cat. No. 48389, 1:100), rabbit polyclonal anti-Pax8 (Proteintech, Cat. No. 10336-1-AP, 1:50), mouse monoclonal anti-acetylated tubulin (Sigma, Cat. No. 6-11-B1, 1:100), rabbit polyclonal anti-LGR6 (Antibodiesonline, Cat. No. ABIN122588, 1:100), mouse monoclonal anti-Ca 125 (Calbiochem, Cat. No. Ca1004, 1:100), mouse monoclonal anti-occludin (Invitrogen, Cat. No. 33-1500, 1:100) and rabbit polyclonal anti-vimentin (Cell Signaling, Cat. No. 5741, 1:100). We further used the following: Draq5 (Thermofisher Scientific, Cat. No. 62251), γ-secretase Inhibitor XX (Calbiochem, Cat. No. CAS 209984-56-5), β-oestradiol (Sigma, Cat. No. E2758) and progesterone (Acros Organics, Cat. No. Cas 57-83-0).

Proteins were extracted from cell cultures and lysed with Laemmli sample buffer containing 2% SDS, 52.5 mM Tris-HCl and protease inhibitors (Roche diagnostics). Equal amounts of protein from each treatment were loaded into 12% SDS-polyacrylamide gels. Anti- EB1 (clone KT51, Abcam), anti-EB2 (Abcam), anti-EB3 (EPR11421(B), Abcam), anti-α-tubulin (Sigma-Aldrich), anti-acetylated tubulin (6-11B-1, Merck millipore), anti-detyrosinated tubulin (Abcam), and anti-GFP (Abcam), anti-GAPDH (Clone, source), anti-acetylated histone H3 (Merck millipore) and anti-mouse IgG-horseradish peroxidase (Jackson Immunoresearch) were used. YL½ antibody (Merck Millipore) was used to detect both tyrosinated tubulin (~ 50 kDa) and tyrosinated EB1 (~ 30 kDa). U87 cells were transfected with GFP-EB1 and Detyrosinated-GFP-EB1 plasmids [19 (link)] using lipofectamineTM 2000 system (Invitrogen) and left to incubator for 24–48 h. Visualization of protein bands was performed with a chemiluminescence detection kit (Millipore) and the chemiluminescent signal was acquired with a G:BOX imaging system (Syngene). Quantification of western blot bands was performed with Image J software.