Figure 3. Pretreatment with proteasome inhibitor, Aclacinomycin, does not modulate PV-induced tyrosine phosphorylation.
(A). ILU-18 cells were treated with PV (100μM) for 2 hours, with or without prior treatment with Acla (0.25μM) for 2 hours. At the end of incubation, cells were washed and cell lysates prepared. As controls, cell lysates were made from ILU-18 cells left untreated or treated for 4 hours with 0.25μM Aclacinomycin alone. Lysates, equalized for protein, were analyzed by immunoblotting using an antibody specific to Phospho-Tyrosine residues. Molecular weights derived from standards are indicated in kDa.
(B). ILU-18 cells were treated with PV (100μM) for 20 min, with or without prior treatment with Aclacinomycin (0.25μM) for 2 hours. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells either left untreated or treated for 140 minutes with 0.25μM Aclacinomycin alone. Lysates, equalized for 30μg protein, were resolved using SDS-PAGE, followed by western blotting using both antibodies to IκBα and phospho-IκBα (Tyr-42) and ECL. After stripping, the blot was reprobed with anti-β-actin to demonstrate equal loading.
