Figure 10. Effect of Kaposin A on phosphorylation of degron mutant REST.

(A) 293T cells were transduced with vector alone or retroviruses expressing FLAG-WT-REST or Kaposin A and FLAG-WT-REST or Kaposin A and FLAG-mutant REST. Upper panel shows cytoplasmic fractions immunoprecipitated with anti-phosphoserine antibody followed by Western blot with anti-FLAG antibody. Middle panel shows cytoplasmic fractions immunoprecipitated with anti-FLAG antibody followed by Western blot with anti-β-TRCP. Bottom panel shows whole cell extracts subjected to Western blot using anti-β-TRCP antibody. (B) 293T cells were transduced with vector alone or Kaposin A for 2 days and the Kaposin A transduced cells were transfected with FLAG tagged REST-WT or FLAG-REST individually mutated at serine 1024, (FLAG-REST S1024A), 1027 (FLAG-REST S1027A), or 1030 (FLAG-REST S1030A) for 48 h. Cell lysates were IPed with anti-phosphoserine antibody followed by immunoblot with anti-FLAG antibody (First panel) or IP-ed with anti-FLAG antibody followed by immunoblot with anti-phosphoserine antibody (Second panel) or immunoprecipitated using anti-FLAG antibody and Western blotted with poly-ubiquitin antibody (Third panel). The fourth and fifth panels show cell lysates subjected to Western blot using anti-FLAG, and anti-β-actin, respectively. (C) Effect of Kaposin A siRNA on REST phosphorylation: 293T cells transduced with vector alone or Kaposin A were transfected with FLAG-REST S1027A or FLAG-REST WT followed by transfection with control siRNA (C siRNA) or a pool of Kaposin A specific siRNA (Kap A siRNA). After 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti- FLAG antibody followed by immunoblot with anti-phosphoserine antibody (First panel). The membrane was striped and reprobed with anti-FLAG antibody as shown in the second panel. Whole cell extracts were subjected to Western blots analysis using anti-Kaposin A for Kaposin A expression (Third panel). β-actin was used as loading control.