Identification of several ISGylation substrates in erythroid cells and increased ubiquitination in ISG15-/- RBCs.
(A) Proliferating erythroblasts were left either untreated (1), or stimulated with IFNß (100 U/ml) for 24 h (3), or induced to differentiate for 72 h (lane 4 to 8). Cells were lyzed according to size and number and ISG15/ISGylation level was compared by western blotting to the level of ISG15/ISGylation present in proliferating Flag-ISG15-expressing p53-/- erythroid cell line. The arrow points to ISG15 band (B) Quantification of ISG15 level in independent western blot experiments. (C) Proliferating control and Flag-ISG15-expressing p53-/- erythroid cell line were lysed and ISGylated proteins searched after a Flag immunoprecipitation. Anti-Flag detect ISG15-Flag and ISGylated proteins (arrow heads) in the crude lysates (dashed line) and are found enriched after the immunoprecipitation (plain line bar). Stars point to light and heavy chains of immunoglobulin. For the detection of ISGylated Globins, cells were induced to differentiate for 72 hours in order to induce globin expression. ISGylated proteins are indicated by arrowheads. Normal unmodified molecular weight of the proteins are: ERK2 (42kDa), PLCγ (150kDa), STAT5 (90kDa), Globin (13kDa). For ERK2 and STAT5 detection, cell lysates were run on a 10% acrylamide gel, for ISG15 and Globins detection on a 15% acrylamide gel and for PLCγ on a 8% gel. (D) Proliferating or 48 hours-differentiating control and STAT5-Flag-expressing p53-/- cell line were lysed and analyzed for exogenous STAT5 expression either using a Flag (upper left panel) or a STAT5 antibody (upper right panel). Note the modest increase in the total amount of STAT5 induced by the expression of STAT5-Flag. ISGylated STAT5 was searched after a Flag immunoprecipitation followed by either a Flag (bottom left panel) or an ISG15 western blot analyses (bottom right panel). Extracts were run on a 7% acrylamide gel. (C) Western blot analysis of RBC extracts from WT and ISG15-/- mice using a 15% acylamide gel. ISG15 expression and ISGylation were analysed using anti-ISG15 antibody (top panel), ubiquitination was monitored using anti-ubi antibody (intermediate panel) and anti-β-Actin was used as a loading control.