Sas4 stimulates Plk4 phosphorylation of Ana2 S38. (A) Phospho-mutations in the STAN domain (3A or 3PM) of Ana2 do not affect the ability of Sas4 and Plk4 to hyperphosphorylate Ana2. S2 cells were cotransfected with the indicated constructs and the next day induced to express for 24 h by addition of 1 mM CuSO₄. Anti-GFP IPs were then prepared from lysates, and Western blots of the inputs and IPs probed for GFP, V5, and α-tubulin. (B) Double phospho-mutation of NT residues T33 and S38 (2A or 2PM) alter the electrophoretic shift of Ana2. S2 cells were treated as in A. Anti-GFP IPs were then prepared from lysates, and Western blots of the inputs and IPs were probed for GFP, myc, V5, and α-tubulin. (C) Phospho-mutations in S38 but not T33 affect the electrophoretic shift of Ana2. S2 cells were treated as in A. Anti-GFP IPs were then prepared from lysates, and Western blots of the inputs and IPs probed for GFP, myc, V5, and α-tubulin. (D and E) The phospho-null mutation S38A and the Sas4-binding mutation P11A/R12A prevent the shift in Ana2’s electrophoretic mobility. S2 cells were Ana2-depleted by RNAi targeting the UTR for 6 d. On day 4, cells were transfected with the indicated constructs and induced to express the next day for 24 h by the addition of 1 mM CuSO₄. Whole cell lysates (D) or anti-GFP IPs (E) were then prepared and Western blots of the samples were probed for GFP, V5, myc, and α-tubulin. (F) Table shows the presence (Yes or No) of NT phospho-residues in Ana2 IPed from cells with the indicated treatments. (G) Coomassie-stained protein gel of immunoprecipitated Ana2 from S2 cells coexpressing Sas4-GFP and either active (ND) or KD Plk4-myc. Gel slices corresponding to the upper and lower migratory species of Ana2 (dashed lines) were isolated and prepared for MS/MS analysis. Detection (yes or no) of phospho-S38 within each gel region by MS/MS is shown.