Fig. 3
Inactivation of STAG2 in primary human cells causes disruption of cohesin interaction with the replication machinery along with failure to establish SMC3 acetylation. a Immunoprecipitation using antibodies against the cohesin subunit SMC3 pulls down multiple components of the pre-replication complex and replication machinery, but not the origin recognition complex, across a spectrum of human cell lines. b Immunoprecipitation using STAG2 antibodies demonstrating interaction of the replication helicase MCM3 with STAG2. c Summary of immunoprecipitation results in HeLa and RPE cells demonstrating a specific interaction of MCM3 with the cohesin subunits STAG2, SMC3, SMC1A, and PDS5A, but not the cohesin regulatory factors NIPBL and Securin. d Immunoprecipitation using SMC3 antibodies of lysates collected from RPE cells following treatment with DMSO vehicle, the CDK4/6 inhibitor palbociclib, the DNA polymerase inhibitor aphidicolin, and the microtubule polymerization inhibitor colcemid. e Immunoprecipitation of lysates from RPE cells using SMC3 antibodies in the presence of DNA nuclease treatment. f shRNA depletion of STAG2 disrupts cohesin interaction with the replication factors PCNA, DNA polymerase epsilon, and DNA polymerase delta, while enhancing the interaction with the single-stranded DNA binding protein RPA/p34, the MCM helicase complex, and replication licensing factors (CDC6, CDT1, and Geminin). g, h Assessment of SMC3 acetylation in RPE cells after lentiviral transduction with empty pLKO.1 or two independent STAG2 shRNAs. Shown are immunoblots using antibodies against acetylated-lysine following SMC3 immunoprecipitation (g), as well as immunoblots of total SMC3 and STAG2 proteins on whole cell lysates (h). Source data are provided as a Source Data file
