Figure 1.

Proteasomal degradation of PIP5Kα by NEDD4. HEK293 cells were treated with lactacystin (L), MG132 (M), chloroquine (C) (each 10 μmol/L), NH ₄Cl (N, 1 mmol/L), or DMSO (D, a vehicle control) for 4 h (A), or with 10 μmol/L MG132 (B) or 10 μmol/L cycloheximide (CHX) (C) for the indicated times. (A‐C) The PIP5Kα protein in cell lysates was analysed by immunoblotting. HEK293 cells were transfected with different amounts of HA‐NEDD4 (D) or with control siRNA or two NEDD4 siRNAs (E), as indicated. HEK293 cells were cotransfected with GFP‐PIP5Kα and HA‐NEDD4 (F) or with the WT or inactive (C867A) HA‐NEDD4 together with FLAG‐PIP5Kα (G). (H) HA‐NEDD4‐transfected cells were left untreated or treated with MG132 (10 μmol/L) for 1 h. (D‐H) Cell lysates were analysed by Western blotting with the indicated antibodies. As a loading control, β‐actin or α‐tubulin was included. (A, E‐G) PIP5Kα levels were normalized to β‐actin levels and quantified relative to those in cells with untreated control (A), control siRNA (E) or without HA‐NEDD4 (F, G). Values in the graphs are presented as the mean ± SEM. *P < .05, **P < .01