Fig. 3.

Mid1 induces ubiquitination and reduces the abundance of Pax6 protein. (A) Blots show levels of Pax6, Mid1, and β-tubulin in HEK293 cells after cotransfection on pax6-flag expression plasmid and either EV or myc-mid1 at different time points (min) on cycloheximide block of protein synthesis. Diagram shows the quantified values of Pax6 protein relative to β-tubulin levels in Western blot. Amounts of Pax6 protein before the cycloheximide treatment are designated as 1 (100%). (B) In vivo ubiquitination of Pax6 was analyzed by transfection of HEK293 cells with plasmids coding for pax6-flag, myc-mid1, and his-ubiquitin as indicated and anti-his Western blot of proteins eluted after an anti-flag-IP (Left). Input of the proteins was verified by Western blot of the corresponding lysates with anti-Myc and anti-Pax6 antibodies, respectively (Right).