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. 2020 Jan 31;477(2):541–555. doi: 10.1042/BCJ20190647

Figure 2. LDM protein is rapidly turned over compared with LSS.

Figure 2.

(A) CHO-LSS-myc cells were transfected with pTK-LDM-V5 plasmid for 24 h and treated with or without 10 µg/ml cycloheximide (CHX) for the indicated time. (B) CHO-LDM-V5 cells were treated with or without 10 µg/ml cycloheximide (CHX) for the indicated time. (C) CHO-7 cells were treated with or without 10 µg/ml cycloheximide (CHX) for the indicated time. (D,E) CHO-7 and CHO-LDM-V5 cells were treated with vehicle (Veh), 5 µM compactin (Statin) or 1 µg/ml 25-hydroxycholesterol (25HC) for 24 h. (D) Columns are representative of endogenous LDM (n = 4) protein levels and red lines are representative of ectopic LDM-V5 (n = 1) protein levels. Protein levels were analysed by Western blotting with myc, V5, endogenous LDM, α-tubulin or vinculin antibodies. Total CYP51A1 mRNA levels were measured using qRT-PCR and normalised to the housekeeping gene PBGD. mRNA levels were normalised to vehicle conditions in the CHO-7 cell line which were set to 1. Data are presented as mean ± SEM from at least three independent experiments (A n = 4, B n = 3–6, C n = 4, D n = 1–4, E n = 3), where * P < 0.05 and ** P < 0.01. Relative protein levels were measured using ImageStudio Lite (version 5.2) and normalised to the vehicle condition which was set to 100% (A-C) or 1.0 (D,E).