A0024

For immunofluorescence staining, cells were fixed with 4% formaldehyde in PBS solution for 15 min, permeabilized with 1% Triton X-100 in PBS solution for 15 min and blocked with 2% bovine serum albumin in PBS solution for 1 h. Thereafter, cells were incubated with the following primary antibodies overnight at 4 °C: anti-MAP 2 (1:500; Sigma, M1406), anti-TUJ1 (1:500; Covance, MRB-435P), anti-TAU (1:200; Dako, A0024) and anti-CTIP2 (1:200; Abcam, ab28448). Primary antibodies were detected using secondary antibodies conjugated to Alexa Fluor 488 (1:500; Molecular Probes) and Alexa Fluor 594 (1:500; Molecular Probes). Cells were washed in PBS and nuclei counterstained with DAPI (Sigma-Aldrich, D9542). Finally, coverslips of growing cells were transferred onto glass slides with mounting medium (Dako, S3023) and imaged immediately using sequential line scanning with a Leica TCS SP5 II inverted confocal microscope.

Frozen Drosophila heads were homogenized in 20 μl 2× Laemmli sample buffer (Sigma‐Aldrich, St. Lois, MO, USA), boiled at 100°C for 10 min, run on 15% SDS‐Page precast gels (Bio‐Rad Laboratories, Hercules, CA, USA) and transferred to nitrocellulose membranes following standard procedures. Equal loading and transfer efficiency were assessed by Ponceau S staining (Sigma‐Aldrich, St. Lois, MO, USA). Membranes were blocked in blocking buffer (PBS, 0.1% tween, and 2% milk) for 5 min followed by overnight incubations with the appropriate primary antibodies diluted in blocking buffer at 4°C. Primary antibodies were Tau (DAKO, A0024), actin (Abcam, ab8227). The next day, membranes were washed three times for 5 min in PBS + 0.1% tween followed by a 2‐hr incubation with HRP‐conjugated secondary antibodies at room temperature. Blots were then washed twice for 5 min with PBS + 0.1% tween, once for 5 min with dH2O and developed with an enhanced chemiluminescent substrate (ThermoFisher Scientific).