Phosphorylation Analysis of Par-1 in S2R+ Cells

Wild type FLAG-tagged Par-1 (isoform RR) or the phosphomutant Par-1^T636A were expressed in S2R+ cells by cotransfection of the corresponding pUAST expression plasmids (Herzmann et al., 2017 (link)) with Actin5C-GAL4. After 72 h, 20 μM 20-hydroxy-ecdysone was added to the medium for 3 h. Cells were harvested in ice-cold PBS and lysed in SDS sample buffer. Lysates were run on 8% gels and blotted with antibodies against phosphorylated Par-1 (phospho-MARK family, #4836; 1:1,000; Cell Signaling Technology). FLAG M2 (F3165; 1:5,000; Sigma-Aldrich) and Drosophila VCP (Rumpf lab, raised against recombinant VCP in rabbits at Pineda antibody service, 1:5,000) as a loading control on an Amersham Imager 680.