Supplementary Figure 3: IMPDH2 protein levels are upregulated in murine and human GBM.

a, Validation of IMPDH1 and IMPDH2 antibodies for their isotype specificity and reactivity in western blot. Of note, upregulation of IMPDH1 in IMPDH2 knockdown cells was observed due to the compensatory mechanism¹. n=1 experiment. b, Validation of IMPDH1 and IMPDH2 antibodies for isotype specificity and reactivity in immunohistochemical (IHC) analysis. IMPDH1 and IMPDH2 immuno-staining in paraffin-embedded IMPDH-knocked down and Flag-IMPDH-overexpressing 293T cells. Each IMPDH1 and IMPDH2 antibody selectively recognizes endogenous IMPDH1 and IMPDH2, respectively and does not react with over-expressed IMPDH2 and IMPDH1, respectively. Of note, endogenous IMPDH1 is upregulated upon IMPDH2 knockdown as seen in (a). n=1 experiment. c, IMPDH2 expression in the Gfap-CreER;Pten^fl/fl;Rb^fl/fl;Tp53^fl/fl;Rbl1^-/- mouse brain analyzed by western blotting (upper, n=4 independent animals) and immunohistochemistry (lower, a representative of 8 independent animals). d, No detectable IMPDH1 protein in the Cohort #4. n=1 for grade 2, n=5 for grade 3, n=30 for grade 4. e, Quantification of cellular IMPDH1 and IMPDH2 protein levels show higher IMPDH2 concentration than IMPDH1 in GBM. His-tagged IMPDH1 and IMPDH2 recombinant proteins—for standard—and cell lysates from GBM cells were subjected to SDS-PAGE. After detection of each IMPDH isoform, cellular IMPDH concentrations were calculated using the standard curves generated by the recombinant IMPDHs and cellular volume as described in the method section. n=1 experiment. f, g, IMPDH2 protein levels are much higher than IMPDH1 in GBM. Western blotting analysis using lysates from GBM cell lines, human and mouse GBM stem cells, primary glia and mouse NSC. Non-tagged and Flag-tagged IMPDHs were used as internal controls to compare the expression levels of IMPDH1 and IMPDH2 in GBM cells. n=1 experiment.