Nedd4

The treated RPE cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton-X100 for 10 min, blocked with 5% BSA for 1 h and incubated with the primary antibody at 4 °C overnight. After washing with PBS, the sections were stained with FITC or Cy3-conjugated goat-anti-rabbit secondary antibody (Sigma-Aldrich, St. Louis, USA) for 1 h at 37 °C, counterstained with Hoechst 33,342 (Sigma-Aldrich, St. Louis, USA) and observed under a confocal microscope (DeltaVision Elite, GE, USA). The primary antibodies included Ki-67 (1:100, CST, 9449), NRF2 (1:100, Bioworld, BS1258), PTEN (1:100, Bioworld, BS1305), p-AKT (1:100, CST, 4060) and NEDD4 (1:100, Proteintech, 21,698–1-AP).

Cells were washed with PBS and then lysed with RIPA buffer. Insoluble materials were removed by centrifugation at 12,000 rpm for 15 min at 4 °C. Equal amounts of protein were separated using 10% SDS-PAGE, transferred to PVDF, and probed with the appropriate antibodies as indicated. Immunoreactive bands were visualized using an ECL kit (Amersham Biosciences, Piscataway, New Jersey, USA). The extraction of proteins from nuclear and cytoplasmic fractions were performed using Subcellular Protein Fractionation Kit from Thermo Scientific (78840; Waltham, MA, USA). Primary antibodies including: NDRG1 (Rabbit, catalog number ab124689) from Abcam; p21 (Rabbit, catalog number 2947) from Cell Signaling Technology; p21 (Mouse, catalog number ab80633) from Abcam; NEDD4 (Rabbit, catalog number 2740) from Cell Signaling Technology; NEDD4 (Rabbit, catalog number 21698-1-AP) from Proteintech; Flag (Rabbit, catalog number 14793) and Histone H3 (Rabbit, catalog number 4499) from Cell Signaling Technology. The secondary antibodies implemented include horseradish peroxidase-conjugated anti-goat (catalog number A5420), anti-rabbit (catalog number A6154), and anti-mouse (catalog number A4416) antibodies from Sigma-Aldrich.

Antibodies against PHB2 (Cat#14085S), PHB1 (Cat#2426S), SHIP2 (Cat#2839S), Akt (Cat#9272S), pAkt-Ser473 (Cat#4060S), pAkt-Thr308 (Cat#13038S), p21 (Cat#2947S), p27 (Cat#3686S), Histone H3 (Cat#9715S), K63 (Cat#5621S), K48 (Cat#8081S), and Ki67 (Cat#34330SF) were purchased from Cell Signaling Technology (Beverly, MA, USA), antibodies against tGFP (Cat#TA150041) was purchased from OriGene (Rockville, MD, USA), antibodies against Flag (Cat#66008-3-Ig, Cat#20543-1-AP), HA (Cat#51064-2-AP, Cat#66006-2-Ig), His (Cat#66005-1-Ig), GST (Cat#66001-2-Ig), Ubiquitin (Cat#10201-2-AP), β-actin (Cat#66009-1-Ig), NEDD4 (Cat#21698-1-AP), SIAH2 (Cat#12651-1-AP), WWP2 (Cat#67274-1-Ig), Cul4A (Cat#14851-1-AP) and MUL1 (Cat#16133-1-AP) were purchased from Proteintech (Wuhan, China). MG132 (Cat#S1748) was purchased from Beyotime Biotechnology (Shanghai, China). Cycloheximide (CHX) solution was purchased from Sigma-Aldrich (St. Louis, MO, USA).