Dephosphorylation of pocket proteins in response to AG1024. Dephosphorylation of pocket proteins in response to AG1024. A, cell extracts from melanoma (YUSAC2) cells were fractionated in 6% precast polyacrylamide gels and Western blotted with antibodies to pocket proteins (pRb, p107, and p130) normalized to a spurious band (control). Serum-deprived melanoma cells were treated with 200 nm AG1024 for the indicated time intervals. B, AG1024 reduces the phosphorylation levels of pRb at Ser608 and Ser780 but not at Ser795 site in a concentration and time-dependent manner. Melanoma cells (501 mel) were serum-starved for 2 days and then treated with AG1024 as indicated. Cell extracts were fractionated in 8% precast polyacrylamide gels and Western blotted with antiphospho-Ser608, antiphospho-Ser780, antiphospho-Ser795, or anti-pRb mAb IF8. C, suppression of pRb phosphorylated forms in response to AG1024 normal melanocytes compared with melanoma cells. Normal melanocytes (NM) were incubated in Ham’s F-10 medium (without serum) supplemented with defined growth factors for 3 h before the addition of AG1024 (1 h, 4 μm). Melanoma cells 501 mel (501), YUSAC2 (SAC), and YUSIT1 (SIT) were serum-starved for 2 days and then treated with AG1024 (1 h, 4 μm). Cell extracts were fractionated in 8% precast polyacrylamide gels and subjected to Western blotting with antiphospho-Ser608, antiphospho-Ser780, antiphospho-Ser807/Ser811, anti-pRb mAb IF8, or antiactin as indicated. Downward pRb band shifts after AG1024 treatments are less apparent in 8% polyacrylamide gels here and in subsequent figures (Fig. 7) <$REFLINK> . Maria von Willebrand et al. Cancer Res 2003;63:1420-1429 ©2003 by American Association for Cancer Research