Figure 5.

Arg-GlcNAcylated FADD is stable and irreversible in response to physiological temperatures. Immunoblots of in vitro generated Arg-GlcNAcylated His-FADD incubated at various temperatures are shown. A, time course of in vitro NleB1-modified His-FADD stored at 4 °C for up to 100 days. B, time course of in vitro NleB1-modified His-FADD stored at 37 °C for 9 days and 21 °C for 15 days. Heat inactivation of NleB1 prior to incubation with His-FADD resulted in the inhibition of Arg GlcNAcylation. Incubated proteins were separated by SDS-PAGE, immunoblotted, and probed with anti-GlcNAc antibody to detect modified FADD. Anti-His, anti-GST, and anti-β-actin antibodies were used for control immunoblots.