Extended Data Fig. 7. Enhanced HOXA10 and IRF8 phosphorylation in myeloid cells during culture with GM-CSF and IL-3 in the presence of anti-PD-L1 blocking antibody.
a, b, Bone marrow cells from C57BL/6 wild-type mice were cultured for 48 hr in the presence of GM-CSF (10 ng/ml) and IL-3 (5 ng/ml), with either IgG control of anti-PD-L1 blocking antibody (MIH5) (10 µg/ml). Cell lysates were prepared and immunoprecipitation was done with agarose-conjugated HOXA10-specific antibody or agarose-conjugated IRF8-specific antibody followed by SDS-PAGE and immunoblot with anti-PY and HOXA10 antibodies or anti-PY and and IRF8 antibodies (a). The abundance of phosphorylated HOXA10 was normalized to immunoprecipitated HOXA10 and the abundance of phosphorylated IRF8 was normalized to immunoprecipitated IRF8 and were expressed as fold change over the relevant values obtained in cells cultured without PD-L1 blocking antibody (defined as 1) (b). Expression of actin in whole cell lysates was also examined as input. Images were visualized, acquired and quantified with Li-COR Odyssey CLx imaging system. Results are from one of three independent experiments. Values of three separate quantifications per condition are shown.