For construction of UAST-GFP-alphaTub84B K40R, (non-acetylatable alphaTub84B) site directed mutagenesis was performed using primers:
5'-GCGGAGGTGATGACTCGTTCAACACCTTC-3',
5'-CCACGGTCCTGTCAGACGGCATCTGGCCAT-3' (K40 site is underlined) from UASP-GFP-alphaTub84B plasmid (gift from Allan Spradling). Resultant plasmid was used to amplify alphaTub84B K40R insert using primers with restriction sites (lowercase):
BglII alphaTub84B-F; 5'-TACagatctATGCGTGAATGTATCTCTATCCATG-3',
NotI alphaTub84B-R; 5'-gcggccgcTTCTGCTATACGTGTCTTTGTGGATAA-3'.
The amplified fragment was subcloned into BglII/NotI sites of pUAST-GFP-attB vector (gift from Cheng-Yu-Lee) and sequenced. Transgenic flies were generated using by PhiC31 integrase-mediated transgenesis (BestGene).