Protein extracts were prepared from heads or dissected brains. Cell homogenization was done in RIPA buffer (150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris, pH 8.0, supplemented with protease inhibitor cocktail from Roche Life Science). After 2 hrs of lysis at room temperature, Laemmli buffer was added before to boil the samples during 5 min.
Samples were analyzed by SDS-PAGE. To separate alpha and beta-tubulins, we used a protocol described by Banerjee and collaborators63 (link). Separated proteins were electrophoretically transferred onto nitrocellulose membrane (Hybond C-Extra, Amersham Biosciences) prior to blotting. Primary and secondary antibodies were incubated in 5% milk in PTX (PBS, 0.1% TritonX100) and washes were done with PTX. Immunodetection was done with Clarity Western ECL kit (BIO-RAD). Chemiluminescence detection was acquired with ChemiDoc Touch Imaging System (BIO-RAD). The following primary antibodies were used: mouse GT335 (1/200, AdipoGen), mouse 1D5 (1/500, Synaptic System), mouse anti-acetylated-tubulin (1/2000, Sigma), mouse DM1A (1/4000, anti-alpha tubulin from Sigma), mouse E7 (1/1000, anti-beta tubulin from DSHB), mouse anti-actin (1/2000, ThermoScientific). Secondary antibody used was HRP-linked goat anti-mouse (1/10000, Jackson ImmunoResearch).
Samples were analyzed by SDS-PAGE. To separate alpha and beta-tubulins, we used a protocol described by Banerjee and collaborators63 (link). Separated proteins were electrophoretically transferred onto nitrocellulose membrane (Hybond C-Extra, Amersham Biosciences) prior to blotting. Primary and secondary antibodies were incubated in 5% milk in PTX (PBS, 0.1% TritonX100) and washes were done with PTX. Immunodetection was done with Clarity Western ECL kit (BIO-RAD). Chemiluminescence detection was acquired with ChemiDoc Touch Imaging System (BIO-RAD). The following primary antibodies were used: mouse GT335 (1/200, AdipoGen), mouse 1D5 (1/500, Synaptic System), mouse anti-acetylated-tubulin (1/2000, Sigma), mouse DM1A (1/4000, anti-alpha tubulin from Sigma), mouse E7 (1/1000, anti-beta tubulin from DSHB), mouse anti-actin (1/2000, ThermoScientific). Secondary antibody used was HRP-linked goat anti-mouse (1/10000, Jackson ImmunoResearch).