Fig. 4

HMGCR regulates drug sensitivity via the E3 ubiquitin ligase SIAH1

(A) Ubibrowser predicted the ubiquitinase of HMGCR. (B) The relative mRNA and protein levels of SIAH1, SYVN1, MARCH1, and MARCH8 in the A549/DDP cell lines with CHOL or PPD. (C) The relative mRNA and protein levels of SIAH1 and HMGCR in the constructed A549/DDP cell lines are stable overexpressing or knocking out SIAH1. (D) Flag- SIAH1 was transfected into A549/DDP cells, and then an IP assay revealed the association between SIAH1 and HMGCR. (E) Immunofluorescence analysis showed the colocalization of SIAH1 and HMGCR in A549/DDP cells. (F) Western blot analysis showed HMGCR protein levels at different time points of actinomycin (CHX, 50 µg/ml) treated A549/DDP cells. (G) A549/DDP cells were treated with or without MG132 (10 µM) for 6 h, and then protein was blotted to show HMGCR protein levels. (H) The plasmids shown were cotransfected into H293T cells, and the ubiquitination status of HMGCR was determined using an immunoprecipitation assay. The experiments were repeated three times. For comparisons, a Student’s t test was performed. ^*P < 0.05, and ^**/##P < 0.01