Fig. 4: SNEP1 enhances LNX1-SuFu interactions and facilitates LNX1-mediated SuFu proteolysis.

A An IP assay of SNEP1 with LNX1. After transfection with the indicated plasmids, HEK-293T cells were harvested for a Co-IP assay with an antibody recognizing Flag-tag, and binding proteins were detected via IB. B An IP assay of SNEP1 with LNX1. HT-29 cell lysates were subjected to a Co-IP assay with the indicated antibodies and protein-A/G beads overnight. Bound proteins were detected via IB. C GST pull-down assay of SNEP1 with LNX1. The prokaryotically expressed GST-SNEP1 was incubated with HEK-293T cell lysates overexpressing Flag-LNX1. The protein complexes pulled down with GST were detected via IB. D LNX1 knockdown increases SuFu protein levels. HEK-293T cells were transfected with different doses of sh-LNX1 construct for 48 h and harvested for IB analysis. E SNEP1-mediated SuFu degradation depends on LNX1. IB analysis of HEK-293T cell lysates that expressed GFP-SNEP1 with LNX1 knockdown or not. F Ectopic SNEP1 increases SuFu-LNX1 interactions. HEK-293T cells were cotransfected with HA-SuFu and Flag-LNX1 with or without GFP-SNEP1 plasmids and treated with MG132 overnight before harvesting. Cell lysates were subjected to a co-IP-IB assay. G SNEP1 is required for the interaction between SuFu and LNX1. HT-29 cells stably expressing LV-shSNEP1 were treated with MG132 overnight before harvest. Cell lysates were subjected to a co-IP-IB assay. H SNEP1 knockdown reverses the repression of the action of LNX1 on SuFu. IB analysis of HEK-293T cell lysates expressing Flag-LNX1 with or without shRNA-SNEP1. I SuFu knockdown promotes translocation of Gli2 into the nucleus. HCT-116 cells were transfected with Flag-SNEP1, Flag-LNX1 or shRNA-SuFu for 48 h and subjected to IB for detection of cytoplasmic and nuclear fractions of Gli2. GAPDH and PARP-1 were used as cytoplasmic and nuclear loading controls, respectively. J Quantification of I using ImageJ software. Data are shown as the mean ± SD, n = 3, **p < 0.01, ***p < 0.001.