Protein extracts were obtained as described in protein extracts paragraph with a complete lysis buffer.
10 µg of proteins were mixed with Laemmli buffer and then denatured at 95 °C for 5 min. Proteins were separated in a 13% acrylamide SDS-PAGE and, after electrophoresis, transferred to nitrocellulose filters for western blot analysis.
Membranes blocking, washing and antibody incubation were performed according to manufacturer’s instructions. Antibodies against β-tubulin (1:2000, Sigma-Aldrich, USA) and GTP-tubulin (1:1000, Adipogen, USA) were used. After incubation with primary antibodies, membranes were washed and then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000) (anti-mouse, Chemicon, USA; anti-rabbit, PerkinElmer, USA; anti-human, Sigma-Aldrich, USA). Immunoreactive proteins were visualized using an ECL chemiluminescence system (Amersham, USA).
10 µg of proteins were mixed with Laemmli buffer and then denatured at 95 °C for 5 min. Proteins were separated in a 13% acrylamide SDS-PAGE and, after electrophoresis, transferred to nitrocellulose filters for western blot analysis.
Membranes blocking, washing and antibody incubation were performed according to manufacturer’s instructions. Antibodies against β-tubulin (1:2000, Sigma-Aldrich, USA) and GTP-tubulin (1:1000, Adipogen, USA) were used. After incubation with primary antibodies, membranes were washed and then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000) (anti-mouse, Chemicon, USA; anti-rabbit, PerkinElmer, USA; anti-human, Sigma-Aldrich, USA). Immunoreactive proteins were visualized using an ECL chemiluminescence system (Amersham, USA).