Figure 4. MPG is a substrate of ATM and phosphorylation of MPG is essential for function.

a. Primary amino acid sequence of MPG across species demonstrates that the pSQ ATM substrate residue is evolutionarily conserved.

b. Immunoblotting of ATM, phospho-ATM (pATM), ATR and phospho-ATR (pATR) in adult GBM (T98G) and pediatric GBM (SJG2, KNS42 and RES259) cell lines in the presence (+) and absence (-) of TMZ. Cells were treated with TMZ for 48h.

c. Immunoblots demonstrating co-immunoprecipitation of ATM and MPG in adult (T98G) and pediatric (SJG2) GBM cell lines. Both proteins are detected following immunoprecipitation (IP) of either ATM or MPG.

d. Immunoblots demonstrating detection of MPG following immunoprecipitation with a phospho-(serine/ threonine) (pSQ) ATM/ATR substrate antibody in both adult (T98G) and pediatric (SJG2) GBM cell lines in the absence (-) but not in the presence (+) of an ATM inhibitor (ATM inb, ku55933 used at 5uM).

e. Denaturing immunoprecipitation of MPG and immunoblotting of pSQ substrate specific antibody in presence and absence of ATM inhibitor to demonstrate MPG is phosphorylated at the SQ residue. W.C.L=whole cell lysate. ATM inhibitor ku55933 was used at 5uM

f. In vitro kinase ATM substrate assay. Immunoblots for thiophosphate ester to detect phosphate incorporation into MPG from a thiol labelled ATP analog in presence of ATM kinase. In vitro kinase assay was performed with wild-type MPG (MPG WT), mutant MPG (MPG S172G) in the presence (+) or absence (-) of cold ATP, ATP_γS, PNBM or with an ATM inhibitor (ku55933, 5uM) demonstrating MPG is directly phosphorylated by ATM kinase at serine 172.

g. Fresh lysed whole cell lysates from e, were used for the MPG molecular beacon assay to confirm that inhibition of ATM resulted in reduced MPG glycosylase activity.