ING1a delays growth factor signaling, delays Rb phosphorylation, and inhibits E2Factivation.

<p>(A) Low passage (young) Hs68 fibroblasts (MPD 25) infected with Ad-GFP or Ad-ING1a for 24 h were serum-starved and stimulated with 100 ng/ml of EGF for the indicated time points. Western blots of p-Src, p-Erk, p-Akt, p-38MAPK, and p-Rb were performed to check their activation status as estimated by their phosphorylation. The same lysates were also probed with antibodies for total Src, Akt, p38MAPK, and Rb. Actin was used as the internal loading control. (B) Hs68 cells synchronized by serum starvation for 16 h were released in the presence of Ad-GFP or Ad-ING1a and checked for the phosphorylation status of Rb at indicated time points. (C) Hs68 cells synchronized by serum starvation for 24 h were released in complete medium in the presence of Ad-GFP or Ad-ING1a. Twenty-four hours later, cells were harvested and lysates were immunoprecipiated using anti-Rb antibody. Immunoprecipitates were electrophoresed and blotted with the indicated antibodies. Levels of E2F, Rb, actin, and ING1a were checked in whole cell lysates. (D) Relative mRNA levels of E2F target genes in cells expressing Ad-GFP or Ad-ING1a. The values were normalized to actin (<i>p</i><0.05). (E) Total RNA from cells transfected with ITSN2 was isolated and the expression of p16, p57^KIP2, and Rb were examined. All values were normalized to levels seen in cells transfected with the same amount of control GFP plasmid, which was also used to monitor and control for transfection efficiency.