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. 2021 Sep 27;11(20):9752–9771. doi: 10.7150/thno.63806

Figure 3.

Figure 3

USP32 impairs SLC35F2 protein stability. (A) Exogenous protein levels of SLC35F2 in HEK293 cells were analyzed upon transfection with increasing concentrations of HA-USP32 along with Myc-SLC35F2. Western blot was performed to determine exogenous SLC35F2 expression. (B) The effect of USP32 on endogenous SLC35F2 protein expression was determined in HeLa cells transfected with increasing concentrations of HA-USP32. Western blot analysis was performed to determine endogenous SLC35F2 protein. (C) HEK293 cells transfected with increasing concentrations of USP32 C743A, along with Myc-SLC35F2 to check the effect of the catalytic mutant of USP32 (USP32 C743A). Western blot analysis was performed with the indicated antibodies. (D) The effect of USP32 C743A on endogenous SLC35F2 protein was analyzed upon transfection with increasing concentrations of USP32 in HeLa cells. Western blot analysis was performed with SLC35F2-specific antibodies. (E) HEK293 cells were co-transfected with Myc-SLC35F2 alone or in combination with USP32 sgRNA1 or USP32 sgRNA2. Western blot analysis was used to determine the exogenous SLC35F2 protein level. The graph in the right panel represents expression of the Myc-SLC35F2 protein. (F) Endogenous SLC35F2 protein levels were measured in the presence of sgRNAs targeting USP32 by western blot analysis in HeLa cells. The graph in the right panel represents endogenous expression of SLC35F2 protein. (G) Validation of single cell-derived USP32 knockout clones in HEK293 cells by western blot analysis. Cell lysates were collected and immunoblotted with respective antibodies to analyze endogenous expression of USP32 and SLC35F2. (H) HEK293_USP32KO cells were transfected with either HA-USP32 or HA-USP32 C743A plasmids to measure the reconstitution effect of USP32 on endogenous SLC35F2 protein by western blot analysis. (I) HEK293 cells were co-transfected with Myc-SLC35F2 alone or in combination with HA-USP32 or in the presence of sgRNAs targeting USP32 to check the reconstitution effect of USP32 on exogenous SLC35F2 expression by western blot analysis. (J) Endogenous interaction between USP32 and SLC35F2 was analyzed in HeLa cells. Cell lysates from HeLa cells were immunoprecipitated with USP32- or SLC35F2-specific antibodies and immunoblotted with indicated antibodies. (K) HeLa cells were subjected to Duolink PLA assay and stained with mentioned antibodies. Scale bar = 25 µm.