Alkaline Phosphatase treatment of nuclear extracts alters the mobility of MIXL1 proteins on SDS-PAGE gel. Expression plasmid pCMV5-MIXL1 was transiently transfected to HEK 293T cells and the transfectants were harvested 60 hours later. 15ug of fresh nuclear extracts were treated with 40u calf intestine alkaline phosphatase (CIAP) in the absence or presence of phosphatase inhibitor sodium orthovanadate at 30°C for 15 min. After CIAP treatment, nuclear extracts with orthovanadate and CIAP reactions were resolved on a NuPAGE gel and transferred to a PVDF membrane. Immunobloting was done with anti-MIXL1-N (1:350 dilution). Anti-MIXL1-N detected 4 species on lane 1 (nuclear extracts with orthovanadate and no CIAP treatment). The band α disappeared on mock reaction (lane 2). CIAP treatment resulted in disappearance of both band α and β (lane 3). In contrast, the addition of phosphatase inhibitor orthovanadate protected the all the 4 species (lane 4).