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

Figure 4.

Figure 4

SLC35F2 undergoes ubiquitination and USP32 promotes its ER-associated degradation. (A) HeLa cells were treated with the indicated concentrations of MG132 for 4 h or with indicated concentrations of lysosomal inhibitor CQ. Western blot analyses were performed with the indicated antibodies. (B) HeLa cells were stimulated with 5 or 10 ng/mL of IL-1β for 10 minutes and lysed to capture polyubiquitinated chains from the cell extracts using TUBEs. (C) Endogenous ubiquitination of SLC35F2 was analyzed by transfecting HeLa cells with HA-ubiquitin. Immunoprecipitated with SLC35F2 antibody followed by immunoblotting with HA to monitor the ubiquitination status of endogenous SLC35F2. (D) HeLa cells were treated with 150 µg/mL of cycloheximide (CHX) for the indicated time points and harvested for western blot analysis with SLC35F2 antibodies. The rate of SLC35F2 protein decay was quantified and graphically represented using ImageJ (bottom panel). (E) HeLa cells transfected with indicated constructs and treated with 5 µM MG132 for 5 h before harvesting. Next, SLC35F2-specific antibodies were used to immunoprecipitated the protein, and specific ubiquitin antibodies were used for immunoblotting to check the polyubiquitination status of endogenous SLC35F2 in the presence and absence of HA-USP32 or HA-USP32 C743A. (F) HEK293 cells were transfected with the indicated constructs. Cells were then lysed and immunoprecipitated with Myc antibody and immunoblotted with Flag antibody. (G-I) HeLa cells were transfected with indicated constructs and incubated for 48 h. Next, 150 µg/mL of CHX was treated for the indicated time intervals and harvested for western blot analysis with the indicated antibodies. The rate of SLC35F2 protein decay was quantified in the presence of USP32 sgRNA2 or USP32 WT or USP32 CA and represented graphically using ImageJ (bottom panel). (J) Western blot analysis of HeLa cells transfected with USP32 and treated with or without MG132. (K) HeLa cells transfected with USP32 and treated with or without MG132 were immunostained with SLC35F2 antibody (Red), calnexin (Green), and DAPI (Blue). Scale bar = 10 µm. (L) HeLa cells were subjected to immunofluorescence analysis using calnexin (ER marker) antibody following Duolink PLA assay to check the location of USP32 interaction with SLC35F2. Scale bar = 25 µm. Bottom panel shows the correlation between USP32-SLC35F2 PLA dots and calnexin intensity derived from the Duolink PLA assay (n = 14). (M) Western blot analysis of HeLa cells transfected with USP32 and treated with or without ES I.