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. 2020 Jan 17;11:379. doi: 10.1038/s41467-019-14231-w

Fig. 2. Ubiquitylation and degradation of Insig-2 requires gp78 and the Cys215 residue.

Fig. 2

a CRL1601 cells were transfected with the plasmid expressing Myc-tagged Insig-2 and indicated siRNAs. Cells were then treated with 100 μM cycloheximide (CHX) for indicated periods and harvested for immunoblotting analysis. b Densitometric analysis (n = 3 independent quantification) of Insig-2 levels in (a). AU arbitrary unit. c Schematic of human Insig-2 protein and various mutants. d, e CRL1601 cells were transfected with plasmids expressing different Insig-2 variants in (c), and then treated without or with 100 μM CHX for 5 h followed by immunoblotting analysis. f CRL1601 cells were transfected with indicated plasmids and siRNAs, and treated with 10 μM MG132 for 5 h. Cells were harvested for ubiquitylation assay. g CRL1601 cells were transfected with indicated plasmids and treated with 10 μM MG132 for 5 h. Cells were harvested and lysed in denaturing IP buffer and subjected to ubiquitylation assay. h CRL1601 cells were transfected with indicated plasmids and treated with 10 μM MG132 for 5 h. Cells were harvested, lysed in denaturing IP buffer followed by incubation with the Ni-NTA beads. Precipitates were treated with (+) or without (−) DTT (75 mM) and β-ME (1.5%) prior to immunoblotting analysis.