Fig. 5: Hypoxia increases NICD1 ubiquitination.
From: Hydroxylation of the NOTCH1 intracellular domain regulates Notch signaling dynamics
A NICD1 NNAA mutant is more ubiquitinated compared to the NICD1 wild-type. PhoenixTM cells were transfected with plasmids encoding for HA-tagged NICD1 wild-type (wt), HA-tagged NICD1 NNAA mutant, or an empty vector as a control (eV). After 6 h of treatment with 20 mM of MG132, protein extracts were prepared and subjected to immunoprecipitation (IP) by performing a TUBE assay. Immunoprecipitates were analyzed by Western blotting (WB) versus HA or GAPDH as a loading control. B DMOG treatment enhances NICD1 ubiquitination in Beko cells. Beko cells were treated for 24 h with 0.5 mM DMOG or DMSO as a control. After 6 h of treatment with 20 mM of MG132, protein extracts were prepared and subjected to immunoprecipitation (IP) by performing a TUBE assay. Immunoprecipitates were analyzed by WB versus NICD1 or GAPDH as a loading control. C The increased ubiquitination of the NICD1 NNAA mutant does not require the C-terminal PEST domain. PhoenixTM cells were transfected with plasmids encoding for FLAG-tagged NICD1 wild-type (wt), FLAG-tagged NICD1 deleted of the OPA and PEST domain (ΔOP), FLAG-tagged NICD1 NNAA mutant, FLAG-tagged NICD1 ΔOP NNAA mutant and/or HA-tagged ubiquitin (HA-Ub). Cells were treated with 20 mM of MG132 for 6 h to block the activity of the proteasome. WCE were subjected to FLAG immunoprecipitation (FLAG-IP) and the immunoprecipitates were analyzed by WB versus FLAG or HA.
