RNAi Reduces Expression and Intracellular Retention of Mutant Cartilage Oligomeric Matrix Protein
Figure 2
shRNAs directed against human COMP effectively reduce steady-state levels of COMP mRNA and protein.
COS-7 cells were stably transfected with shRNAs directed against COMP, and individual clones identified and selected as described in Methods section. Cells were infected with an adenovirus expressing MT-COMP and collected 48 hours after infection. RNA and protein were purified for subsequent analysis. (A) Northern blot analysis of steady-state COMP mRNA in transfected cells. (B) Quantification of mRNA in a. Absence of a shRNA was set to 100% and all other lanes were compared to the no shRNA reference sample. snU6 mRNA was used to normalize protein loading. (C) Western blot analysis. (D) Quantification of protein in c in which the level of COMP protein from COS-7 cells expressing recombinant COMP protein in the absence of a shRNA was set to 100% and all other protein levels were compared to this reference. β-actin was used to control for protein loading. All experiments were replicated at least three times.