Stain free 4 15 gels

RIPA supernatants (10 µl) were subjected to electrophoresis on stain free 4–15% gels (Bio-Rad) and then quickly transferred to nitrocellulose using Trans-Blot Turbo Transfer System (Bio-Rad). Proteins on the membrane were revealed using specific antibodies against different forms of modified tubulin (tyrosinated, detyrosinated, Δ2) and α tubulin. Anti-Tyr-Tub (1/10 000), anti-deTyr-Tub (1/20 000), anti Δ2-Tub (1/20 000) and anti α tubulin (1/10 000) antibodies were used with the appropriate peroxidase-/labelled secondary antibodies. Secondary antibody signal was revealed using Pierce ECL western blotting substrate (Thermo Scientific) and analysed with ChemiDoc™MP Imaging System (Bio-Rad) using Image Lab software (stain free gel protocol) for quantification. For each lane of the blot, the software measures the integrated volume of the band corresponding to the antigen of interest. The signal is then normalized according to the total protein measured in the same lane. For every blot, one lane is dedicated to an internal standard corresponding to a wild-type sample (used for the entire study) and the protein-normalized signal of this standard is considered as 100%, therefore each unknown sample is calculated as a percentage of this standard. For each brain sample, three independent blots were performed and the mean intensity was calculated.

RIPA supernatants (10 µl) were subjected to electrophoresis on stain free 4%–15% gels (Bio Rad) and then quickly transferred to Nitrocellulose using Trans-Blot Turbo Transfer System (Bio Rad). Proteins on the membrane were revealed using specific antibodies against acetylated and α tubulin. Anti Acetylated-Tub (1/10,000) and anti α-tubulin (1/10,000) antibodies were used with the appropriate peroxidase-/labeled secondary antibodies. Secondary antibody signal was revealed using Pierce ECL Western blotting substrate (Thermo scientific) and analyzed with ChemiDoc™MP Imaging System (Bio Rad) using Image Lab software (stain free gel and chemoluminescence protocol) for quantification. For each lane of the blot, the software measures the integrated volume of the band corresponding to the antigen of interest. The signal is then normalized according to the total protein measured in the same lane. For every blot, one lane is dedicated to an internal standard corresponding to a WT sample (used for the entire study) and the protein-normalized signal of this standard is considered as 100%, therefore each unknown sample is calculated as a % of this standard. For each brain sample, three independent blots were performed and the mean intensity was calculated.