Fig. 2. Maintenance of Myc levels in ES cells requires suppression of T58 phosphorylation. (A) Top panel: whole cell lysates (40 µg total protein) from LIF maintained ES cells and embryoid bodies (-LIF; see A) were immunoblotted and probed with antibodies raised against Myc, phospho-T58 Myc (Myc^pT58) and GSK3ß; middle panel, extracts as in top panel were probed with anti-Oct4, and GSK3ß antibodies. GSK3ß kinase activity at corresponding times is shown. Bottom panel: quantitation of GSK3ß kinase activity in LIF-maintained ES cells and during differentiation was performed by phosphorimaging analysis and is depicted as a fold increase over initial levels in ES cells. Specificity of the assay was determined by immunoprecipitating GSK3ß from day 6 EB cell lysates and performing kinase reactions in the presence of MBP, GSK3 II inhibitor, ethanol as indicated by +. The presence of the GSK3ß antibody in the immunoprecipitation is as indicated (+). (B) ES cells maintained in LIF were treated with cycloheximide (+CHX, 10 µM) and at 30 minute intervals cells were harvested and whole cell lysates prepared. Following immunoblotting, extracts were probed with antibodies as indicated. Levels of Myc (white circles), Cdk2 (black squares) and cyclin D3 (black circles) are shown for each time point relative to pre-cycloheximide chase levels (t=0). (C) Whole-cell lysates were prepared from mycER LIF-maintained ES cells and day 1, day 2, day 3 and day 4 EBs (-LIF) that had been treated with proteosome inibitor MG132 (+, 5 µM) or ethanol alone (-) 3 hours prior to harvesting. After SDS PAGE and electroblotting onto a membrane, cell lysates (40 µg total protein) were then probed with antibodies raised against Myc and HDAC1/2. (D) Mutation of T58 (T58A) delays the downregulation of Myc protein following LIF withdrawal. mycER or myc^T58AER levels were evaluated in LIF-maintained lines or in EBs generated by growth in the absence of LIF over 5 days (1-5) by immunoblot analysis using anti-Myc and anti-HDAC1 antibodies (loading control). (E) ES cells maintained in Wnt3a CM for five passages (15 days) were harvested or grown as EBs in the absence of CM for 1 or 7 days. Cell lysates were probed with antibodies as indicated and GSK3ß kinase assays performed as described in A but on extracts made from day 7 EBs. The specificity of kinase assays was confirmed as described in A.