JCS028654 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A) The secretion of CypB in CypB-overexpressed cells and knockdown cells. For the immunoprecipitation of secreted CypB, 6 ml of medium collected from the pcDNA-, CypB/wt-, CypB/R62A- or CypB-siRNA-transfected cells was centrifuged and immunoprecipitated in the tubes containing anti-CypB antibody, then analyzed by western blotting. Immunoprecipitation of fresh media (lane 3) without CypB antibody was employed as a negative control. Intracellular CypB was immunoprecipitated from 0.1 mg out of total 2.58 mg cell lysate in pcDNA cells (lane 1). The numbers shown in the left panel were obtained from the densitometric analysis of at least three independent experiments. (B) The defensive role of CypB in the presence of antibody neutralization of secreted CypB. The secreted CypB was neutralized by the application of anti-CypB antibody 2 µg/ml; concentration based on neutralization dose (ND₅₀) for this antibody as defined by the manufacturer) to each of the transfectants (pcDNA, CypB/wt, CypB/R62A, or CypB-siRNA prior to Tg or Tm exposure. The cell survival rate was measured by MTT assay. The data are expressed as the means ± s.d. obtained from five independent experiments. *P<0.05 versus Tg-treated pcDNA; #P<0.05 versus Tm-treated pcDNA.