Figure 7.

K18 depletion does not dampen mTOR/S6K signaling, global protein translation and receptor degradation. (A) Activation of mTOR/S6K signaling pathway in K18 (K18-si) depleted HeLa cells was assessed by immunoblotting whole cell extracts against phosphorylated S6 (S6(P)), total S6, cMet, K18, and Actin as loading control. Immunoblot representative of three different experiments. (B) Rate of total protein synthesis was assessed by ³⁵S-methionine incorporation of HeLa cells transfected with control (Ctr) or K18 targeting (K18-si) siRNA. Autoradiography representative of two independent experiments. (C) Depletion efficiency of the samples that were used for the ³⁵S-methionine incorporation assay. (D) After transfection with control (Ctr) or siRNA targeting K18 (K18-si), HeLa cells were incubated with 100 nM of the lysosomal inhibitor Concanamycin A alone (upper panel) or together with the proteasomal inhibitor 10 μM MG132 (lower panel) for different periods of time. Lysates were collected and immunoblotted for cMet, TfR, integrin β1, K18, and Actin as a loading control. Immunoblots are representative of at least two independent experiments.