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. 2021 Feb 19;12(2):199. doi: 10.1038/s41419-021-03487-0

Fig. 5. SNEP1 facilitates LNX1-mediated ubiquitination of SuFu.

Fig. 5

A SNEP1 knockdown impairs LNX1-mediated ubiquitination of SuFu. HEK-293T cells were transfected with Flag-LNX1, shRNA control, or shSNEP1 for 48 h and treated with MG132 overnight before harvest. Cell lysates were subjected to IP with anti-SuFu antibody and protein A/G beads overnight. Polyubiquitin chains bound to SuFu were assessed via IB. B SNEP1 increases LNX1-mediated ubiquitination of SuFu. HEK-293T cells were transfected with Flag-LNX1, GFP-vector, or GFP-SNEP1 for 48 h and treated with MG132 overnight before harvest. Cell lysates were subjected to IP with anti-SuFu antibody and protein A/G beads overnight. Polyubiquitin chains bound to SuFu were assessed via IB. C SNEP1 enhances LNX1-mediated SuFu ubiquitination in vitro. GST-SuFu, GST-LNX1 (1-600), and SNEP1 were expressed in and purified from E. coli. Purified proteins were mixed with E1, E2, and ubiquitin purchased from Enzo and incubated at 37 °C for 4 h. Polyubiquitin chains were assessed via IB. D The SNEP1-LNX1 complex targets K59 and K470 for SuFu ubiquitination. HEK-293T cells were transfected with GFP-SuFu, GFP-SuFu (K59R), GFP-SuFu (K470R), or GFP-SuFu (K59/470R) and Flag-LNX1 and treated with MG132 overnight before harvest. Cell lysates were subjected to IP with an anti-SuFu antibody and protein A/G beads overnight. Polyubiquitin chains bound to SuFu were assessed via IB. E SuFu (K59/470R) is resistant to degradation mediated by SNEP1. HEK-293T cells were transfected with GFP-SuFu or GFP-SuFu (K59/470R) and Flag-vector or SNEP1 for 48 h before harvest. Cell lysates were assessed via IB. F SuFu (K59/470R) displays a prolonged half-life in cells. CHX (100 μg/ml) was incubated for different time points with GFP-SuFu or GFP-SuFu (K59/470R) and Flag-SNEP1-transfected HEK-293T cells. Cell lysates were harvested for IB with the indicated antibodies. G Quantitative analysis of SuFu protein levels shown in F using ImageJ software. H SuFu (K59/470R) is inactivated in response to LNX1-promoted cell proliferation. HT-29 cells transfected with GFP-SuFu or GFP-SuFu (K59/470R) were treated with EdU for 4 h before fixation with paraformaldehyde. Cells were then stained with rhodamine-tagged anti-EdU antibody and DAPI. I Quantitative analysis of the ratio of EdU+ cells shown in H, **p < 0.01.