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. Author manuscript; available in PMC: 2022 Feb 23.
Published in final edited form as: ACS Chem Biol. 2021 Dec 23;17(1):68–76. doi: 10.1021/acschembio.1c00632

Figure 5.

Figure 5.

Akt phosphorylates NEDD4L (A) Western blot analysis of kinase assays using 100 nM pSer473 Akt (A1) and 1 μM NEDD4L. Assays were carried out using 2 mM ATP at 30 °C for 20 min 200 ng of NEDD4L was loaded per well. Membranes were blotted with the phospho Akt substrate antibody (R/KXR/KXXS*/T*) (1:1000), total Akt antibody (1:5000), NEDD4L, and phospho S342 and phospho S448 (1:2000). (B) Western blot analysis of HCT116 Akt1/2 KO cells transfected with either WT (left) or D274A (catalytically dead, right) Akt and NEDD4L (C942S) showing three different replicates. Anti-Akt(1:2000), pS342 NEDD4L (1:2000), NEDD4L (1:2000), pS448 NEDD4L (1:3000), and anti-GAPDH (1:5000). (C) Bar plot represents quantification of band intensity of WB for phospho NEDD4L forms normalized to total NEDD4L for each band. Data represent four biological replicates with n = 3 for each. P-values are 0.0037 and 0.0063, respectively.