Peroxydase conjugated secondary antibodies

Cells were lysed after 6 h treatment in RIPA buffer (Tris-HCl 50 mM pH 8.0, NaCl 250 mM, Triton-X100 0.1%) with a cocktail of proteases and phosphatases inhibitors (Sigma-Aldrich) added freshly. Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad laboratories, Marnes-la-Coquette, France). Thirty micrograms of total proteins were loaded into a 12% SDS-PAGE gel and electrotransferred onto a nitrocellulose membrane. Primary antibodies used were directed against EB1 (clone 5; BD Biosciences, San Jose, CA), ATP1-A1 (Proteintech Europe, Manchester, UK), Ser 9 phospho-GSK3β (Cell Signaling, Boston, USA), total GSK3β (Thermo Fisher Scientific) and GAPDH (Sigma-Aldrich). Peroxydase-conjugated secondary antibodies (Jackson Immunoresearch, Baltimore, USA) and chemiluminescence detection kit (Millipore, Molsheim, France) was used for visualization of protein bands. Chemiluminescent signal was acquired on a G:BOX imaging system (Syngene, Cambridge, UK) and quantification was done with Image J software.

Proteins were extracted from adherent cells and separated as previously described^{18 (link)}. Primary antibodies were as following: MGMT clone MT3.1 (#NB 100–692 Novus, MO) and anti-actin (clone C4, MAB1501 Millipore). Peroxydase-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, PA) were detected using the ECL detection system (Amersham Biosciences, NJ) (N = 3).

Cells were lysed after 6 hours treatment in RIPA buffer (Tris-HCl 50mM pH 8.0, NaCl 250mM, Triton-X100 0.1 %) with a cocktail of proteases and phosphatases inhibitors (Sigma-Aldrich) added freshly. Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad laboratories, France). Proteins were separated by SDS-PAGE and electrotransferred onto a nitrocellulose membrane. Primary antibodies used were directed against EB1 (clone 5; BD Biosciences), α-tubulin (clone DM1A, Sigma Aldrich), Ser 473 phospho-Akt, total Akt, Ser 9 phospho-GSK3β (Cell Signaling, Boston, USA), total GSK3β (Life Technologies). Peroxydase-conjugated secondary antibodies (Jackson Immunoresearch, Baltimore, USA) and chemiluminescence detection kit (Millipore) were used for visualization, and signal quantification was done with Image J software.