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. 2015 Jul 28;5:12600. doi: 10.1038/srep12600

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(A) NEDD4-1 was knocked down using shRNAs in HEK293 cells expressing the indicated forms of PTEN-GFP (middle images). mCherry-NEDD4-1 was overexpressed together with HA-ubiquitin in the same set of cells (bottom panels). Cells were observed by fluorescence microscopy. Bar, 10 μm. (B) Whole-cell lysates were analyzed using immunoblotting with antibodies to NEDD4-1, actin and GFP to detect the indicated PTEN-GFP fusions. (C) Band intensity was quantified. Values represent the mean ± SD (n = 3). (D) Co-immunoprecipitation of NEDD4-1 and PTEN variants. HEK293 cells expressing HA-NEDD4-1 along with the indicated forms of PTEN-GFP were lysed and incubated with anti-GFP antibodies. Cell lysates (input) and immunoprecipitated fractions (IP) were analyzed by immunoblotting with antibodies to HA and GFP. (E) Band intensity was quantified. Values represent the mean ± SD (n = 3).