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. 2020 Oct 8;8:582695. doi: 10.3389/fcell.2020.582695

FIGURE 7.

FIGURE 7

STK35 is ubiquitinated by NEDD4L and inhibits NEDD4L-mediated anti-colorectal cancer function. (A) Purification of the STK35 immunocomplex. Proteins were separated by SDS-PAGE and stained with Coomassie Blue. (B) Immunoprecipitation was performed with an IgG control, anti-STK35, or anti-NEDD4L antibody, followed by incubation with indicated antibodies. (C) Protein and mRNA expression of STK35 in SW620 cells transduced with NEDD4L-overexpressing lentivirus (NEDD4L) or blank lentivirus (Vector) in the presence of 10 μM MG132 or control (Vehicle), measured with western blot and Q-PCR. β-Actin as the loading control. (D) NEDD4L-overexpressed SW620 cells immune-precipitated with STK35 or IgG antibody for evaluating ubiquitination. (E–I) SW620 cells were transduced with STK35-overexpressing lentivirus (STK35) after NEDD4L-overexpressing lentivirus (NEDD4L) or blank lentivirus (Vector) transfection. (E) Cell apoptosis assessed by flow cytometry and quantification accordingly. (F) Cellular LDH levels measured by biochemical analysis. (G,H) Cellular energy metabolism reflected by (G) oxygen consumption rate and (H) extracellular acidification rate. (I,J) Cellular protein levels of STK35, cleaved caspase-3, cleaved caspase-9, GLUT1, HK-2, p-AKT, and AKT, measured with western blot. β-Actin as the loading control. *** P < 0.001, compared with Vector. ### P < 0.001, compared with NEDD4L.