GAS2L1 undergoes cell cycle-dependent phosphorylation. (A) HeLa cells were released from double-thymidine block into medium containing 5 µM STLC. Whole-cell extracts were collected at indicated times and immunoblotted with antibodies against GAS2L1, cyclin B1, and β-actin. (B) Following double-thymidine release for 12 h, HeLa cells were lysed and incubated at 30°C with or without calf intestinal alkaline phosphatase for 15 min; subsequently, cell extracts were immunoblotted with antibodies against GAS2L1 and β-actin. (C) Left: Preparation of GAS2L1 samples for identification of phosphorylation sites by using mass spectrometry. Right: Coomassie Blue–stained gel of GAS2L1 proteins immunoprecipitated (using the attached tag) from interphase and mitotic cells. (D) Left: GAS2L1 residues identified to undergo mitotically induced phosphorylation. Right: Schematic structure of GAS2L1 indicating the location of five clustered Ser-phosphorylation sites. (E) Sequence alignment of GAS2L1 from various vertebrate species. Asterisks: identified phosphorylation sites within the region.