Fig. 1.

Generation of PCSK9_55 in cells culture. (A) Extracts and conditioned media of HEK293T cells transfected with empty vector (lanes 2 and 5) or with pPCSK9 62 (lanes 3 and 6) were separated via SDS-PAGE and immunoblotted for PCSK9 and actin (MW ladder; lanes 1 and 4). Image is representative of 3 independent experiments. (B) Total PCSK9 quantified in the intracellular and extracellular compartments of our in vitro conditions in HEK293T cells transfected with pPCSK9 62. (C) Experimental strategy followed in cell culture to generate PCSK9_55 under semiphysiologic conditions cotransfecting pPCSK9 62 and pFURIN. (D) Extracts and conditioned media from HEK293T cells transfected with pPCSK9 62 and empty vector (lanes 2 and 4) or pPCSK9 62 and pFURIN (lanes 3 and 5) were separated on an SDS-PAGE and immunoblotted for PCSK9 and actin (MW ladder; lane 1). (E) Quantification of PCSK9 bands from “D” are plotted relative to pPCSK9 62 in the cell extracts and cell media. Image is representative of ≥13 independent experiments. Values are mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (∗) Cell media is loaded with unequal protein load as in cell extracts. Dotted lines indicate splicing of original image.