Anti phospho rps6

Affinity purified polyclonal anti-Cyclin Dd (GDVDLKKFADHLSIPFEFLRC) [18] (link), anti-CKIa (CPRSNLDTKVQKSAIKKS), and anti-E2F1 (SPSLFPSNVANQSVKMSK) antibodies were produced by 21^st Century Biochemical (Malbora MA). Primary antibodies anti-phospho 4EBP1, anti-phospho RPS6, anti-phospho MSK1, anti-phospho p38 (all from Cell Signaling), anti-phospho ERK1/2 (Millipore), anti-Histone H3 and, anti-phospho H3pS28 (Abcam), and anti-IdU and BrdU (Accurate Chemicals) were used for immunofluorescence staining and western blot analysis as described below.

Protein extracts were prepared as previously described (Millen et al., 2009 (link)). Briefly, cells were lysed on ice by resuspension in 1 ml cold H₂O supplemented with 150 µl 1.85 M NaOH and 7.5% (v/v) β-mercaptoethanol. After a 10-min incubation on ice, the protein was precipitated by addition of 150 µl 50% (w/v) trichloroacetic acid. Pellets were washed twice with acetone, resuspended in 100 µl 1× SDS-PAGE buffer, and boiled for 5 min at 95°C. Primary antibodies were incubated overnight at 4°C and were as follows: anti-GFP (1:1000, ab290, Abcam), anti-PGK1 (1:1000, ab113687, Abcam), anti-Rps6 (1:1000, ab40820, Abcam), anti-phospho-Rps6 (1:1000, 4858, Cell Signaling Technology, Danvers). Secondary antibodies were incubated for 1 h at room temperature and were as follows: IRDye 680RD goat anti-rabbit-IgG antibody (926-68171, Li-Cor, Lincoln) and IRDye 680RD goat anti-mouse-IgG antibody (926-68070, Li-Cor). These were detected using the ChemiDoc MP Imaging System (Bio-Rad). Bands were integrated and quantified using Fiji. See Fig. S7 for original blots.