Effect of Asb-4 on protein levels and ubiquitination of IRS4. A.HEK293 cells were transfected with 1 μg (35 mm dish) Flag-IRS4 vector and differing amounts of Myc-Asb-4 or Myc-Asb-4/Δsb vector. The total amount of DNA in each transfection was equalized with pCMV empty vector. Cell lysates were first blotted with anti Flag antibody. The membranes were then stripped and reblotted with anti-Myc and anti-β-actin antibodies, respectively. B. Densitometry analysis of three separate experiments (n = 3). *p < 0.05 vs. control. C. HEK cells were transfected with Myc-Asb-4 vector and the total amount of DNA in each transfection was equalized with empty vector. D. Densitometry analysis of three separate experiments (n = 3). *p < 0.05 vs. control. E. Ubiquitination of IRS4 by Asb-4. CHO cells were co-transfected with Myc-ubiquitin, Flag-IRS4 and empty vector (Lane 1), or Myc-Asb-4 (Lane 2), or Myc-Asb-4/Δsb (Lane 3). Cell lysates were immunoprecipitated with anti-Flag conjugated agarose and first blotted with anti-Myc (top panel), and then the membrane is stripped and blotted with and Flag antibody (middle panel). The cell lysates were blotted with anti Myc antibody to show the expression of Myc-Asb-4 and Myc-Asb-4/Δsb (bottom panel).