The effects of PPP on IGF-1R RTK autophosphorylation and downstream signaling proteins. (A) IGF-1R was extracted from total cell lysates of the RPMI 8226 and the Karpas 707 cell lines and incubated with PPP at indicated concentrations before the kinase reaction was initiated by the addition of ATP. The autophosphorylation of the IGF-1 RTK was quantified spectrophotometrically. ♦ indicates RPMI 8226; ▪, Karpas 707. (B) Insulin R was extracted from total cell lysates of the RPMI 8226 and Karpas 707 cell lines and incubated with PPP at indicated concentrations before kinase reaction was initiated by the addition of ATP. The autophosphorylation of the insulin R was quantified spectrophotometrically. ♦ indicates RPMI 8226; ▪, Karpas 707. (C) Expression of p-Erk1/2, tot-Erk1/2, and the cell-cycle–associated p-CDK1, tot-CDK1, cyclin B1, cyclin D2, and cyclin E was analyzed by Western blotting of serum-starved RPMI 8226 cells incubated for 2, 4, and 24 hours with or without 1 μM PPP. (D) Similarly, expression of p-CDK1 and tot-CDK1 was analyzed in the mouse MM cell line 5T33MMvt lacking the IGF-1R. (E) Expression of p-Akt, tot-Akt, and the downstream signaling proteins GSK-3β p70S6K and their corresponding phosphorylated forms were analyzed according to the same procedure. Protein expression of (F) bcl-x_L, mcl-1, survivin, and (G) hsp60, hsp70, and hsp90 was analyzed by Western blotting of serum-starved RPMI 8226 cells incubated for 2, 4, and 24 hours with or without 1 μM PPP. In all experiments the cells were stimulated with 6.7 nM IGF-1 for 5 minutes at each time point.