Fig. 4

Degradation of ubiquitinated SNIP1 by MKRN1 requires E3 ligase activity. A Degradation of SNIP1 by MKRN1. Expression vectors for Flag-MKRN1 (0, 1, 2, 4, and 6 µg) were introduced into HCT15 cells; steady-state expression levels of SNIP1 and MKRN1 were determined by WB. B Expression vectors containing Flag-SNIP1 (1 µg) and Flag-MKRN1 (1, 2, 4, and 6 µg) were introduced into HCT15 cells; steady-state expression levels of SNIP1 and MKRN1 were detected using WB. C, D MKRN1 affects half-life of SNIP1. HCT116 cells (Control and sh1-MKRN1) and HCT15 cells (vector and OE-MKRN1) were treated with 50 µg/mL cycloheximide and analysed by WB. E, F Effects of MKRN1 on SNIP1 mRNA by quantitative real-time PCR. G Proteasome-dependent degradation of SNIP1 by MKRN1 in HCT15 cells overexpressing MKRN1 and cellular SNIP1 expression after 6 h treatment with 10 µM MG132. H, I MKRN1 mediates SNIP1 ubiquitination. In HCT116 and HCT15 cells, SNIP1 ubiquitination levels after MKRN1 knockdown and overexpression were detected by WB. J In HCT15 cells, the SNIP1 ubiquitination level was measured by WB after a 6-h treatment with 10 µM MG132. K Schematic diagram of the MKRN1 structural domain. L Construction of the E3 ligase mutant H307E of MKRN1. M Validation of MKRN1 (H307E) interactions with SNIP1 using co-immunoprecipitation and WB in HCT15 (H307E) cells. N WB analysis of the effect of the MKRN1 (H307E) mutant on SNIP1 degradation. O SNIP1 degradation by MKRN1 (H307E). Expression vectors for MKRN1 (H307E) (0, 1, 2, 4, and 6 µg) were introduced into HCT15 cells; WB detected steady-state expression levels of SNIP1 and MKRN1. P Effect of MKRN1 (H307E) on half-life of SNIP1. HCT15 cells (vector and MKRN1 (H307E)) were treated with 50 µg/mL cycloheximide and analysed by WB. Q In HCT15 cells (vector, OE-MKRN1, MKRN1 (H307E)), SNIP1 ubiquitination levels were detected by WB after a 6-h treatment with 10 µM MG132