Western Blot Analysis of Drosophila Tau

Heads from adult flies were collected and homogenized in lysis buffer (10 mM Tris–HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 5 mM EGTA; 10% glycerol; 50 mM NaF; 1 mM Na₃VO_4,; 5 mM NaPPi; 5 mM DTT; 4 M urea with protease inhibitor cocktail) at 4 °C. Protein extracts were centrifuged at 3,000 g for 3 min at 4 °C, and the supernatants were stored at -20 °C. Lambda phosphatase (New England Biolabs) was added to the thawed protein extracts and incubated at 30 °C for 30 min. Western blotting was performed following the standard procedure^{45 (link)}. The primary antibodies were used with the following dilutions: rabbit anti-human pan tau (Dako, 1:20,000), mouse anti-tau-C3 (Invitrogen, 1:10,000), mouse anti-α-tubulin (GeneTex, 1:5,000), mouse anti-β-tubulin (Developmental Studies Hybridoma Bank, 1:5,000), mouse anti-AT8 (Thermo, 1:500), mouse anti-AT100 (Thermo, 1:500), mouse anti-ATP5a (Abcam, 1:100,000), rabbit-histone H3 (Abcam, 1:5,000), and mouse anti-syntaxin (Developmental Studies Hybridoma Bank, 1:5,000). Secondary antibodies conjugated with HRP (Jackson ImmunoResearch Laboratories) were used in 1:10,000 dilutions. All loading controls were prepared by stripping off the reagents from the original membrane and then re-immunoblotting following the standard procedures. Semiquantitative analysis of band density was performed in ImageJ.