Ski down-regulation in Schwann cells prevents TGFβ-induced proliferation and ^ser780pRb localization in the cytoplasm. (A and C) Western blot analysis of Ski expression (A) and immunostaining of Ski (red) and DAPI (blue) labeling in Schwann cells infected with a control shRNA (control) or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and kept in growing medium (for Western blot analysis) or treated with TGFβ (for immunostaining; C). (B) Graph representing the percentage of BrdU-labeled rat Schwann cells infected with a control shRNA or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and cultured in DM or treated with TGFβ for 24 h. (D) Immunostaining of ^ser780pRb (pRb; green) and DAPI (blue) labeling (pRb appears turquoise when nuclear) in rat Schwann cells infected with a control or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and treated with TGFβ for 24 h. (E) Western blot analysis of P0 in rat Schwann cells infected with a control or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and cultured in DM. Lysates of cells infected with the control shRNA and the Ski-specific shRNA lentiviruses have been run on the same gel but not on consecutive lanes. (F) Western blot analysis of P0 in rat Schwann cells infected with a Ski-specific shRNA (Ski-0%) and cultured in DM or treated with TGFβ or dbCAMP for 24 h. For A, E, and F, β-actin was used as loading control, and the graphs represent the densitometry of the bands of the protein of interest normalized to the loading control. Statistical analyses were performed using two-tailed t tests on at least three independent experiments. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.