Fig. 5
circINSIG1-121 recruits CUL5-ASB6 complex to promote K48-linked ubiquitination of INSIG1. A Western blot analysis of INSIG1 ubiquitin levels after immunoprecipitation using INSIG1 antibody in HCT8 cells transfected with HA-tagged wildtype (WT) Ub or different ubiquitin chain vectors (K0, K6, K11, K27, K29, K33, K48, K63). B Western blot analysis of K48-linked ubiquitin levels of INSIG1 after immunoprecipitation using INSIG1 antibody in HCT8 cells transfected with circINSIG1 or circINSIG1-121 along with HA-K48-Ub vector. C and D Western blot analysis of INSIG1 and circINSIG1-121 expression in HCT8 cells transfected with circINSIG1 and indicated mutants. E Western blot analysis of INSIG1 and circINSIG1-121 expression in HCT8 cells transfected with circINSIG1 and indicated mutants. F Western blot analysis of INSIG1 and circINSIG1-121 expression in HCT8 cells transfected with circINSIG1 and indicated mutants. G Western blot analysis of K48-linked ubiquitin levels of INSIG1 after immunoprecipitation using INSIG1 antibody in HCT8 cells transfected with circINSIG1 and indicated mutants along with HA-K48-Ub vector. H Western blot analysis of the samples immunoprecipitated by circINSIG1-121 antibody in HCT8 cells. I Immunoprecipitation assay was performed in ASB6-Flag transfected HCT8 cells using Flag antibody, followed by Western blot analysis. J Western blot analysis of INSIG1 expression in HCT8 cells treated with MG132 (10 µM) or MLN4924 (10 µM). K Western blot analysis of K48-linked ubiquitin levels of INSIG1 after immunoprecipitation using INSIG1 antibody in HCT8 cells with ASB6 overexpression or knockdown. L Free cholesterol content detection in HCT8 cells with ASB6 overexpression or knockdown. Values are represented as mean ± SD. ***p < 0.001, by one-way ANOVA