Journal: Frontiers in Physiology

Article Title: Pathogenic gain-of-function mutations in the prodomain and C-terminal domain of PCSK9 inhibit LDL binding

doi: 10.3389/fphys.2022.960272

Figure Lengend Snippet: Ser-127 residue is critical to LDL binding function of PCSK9. (A) 24 h post-transfection, HEK293 cells transiently expressing WT-PCSK9 or indicated S127 mutants were harvested and cell lysates and medium immunoprecipitates were analyzed by 8% SDS-PAGE and western blotting to detect precursor (P) and mature (M) forms of PCSK9. # indicates a faster-migrating form of PCSK9 consistent with furin-mediated proteolysis. Shown is a representative experiment ( n = 3) (B) Mouse Hepa-1c1c7 cells cultured in sterol-depleting medium were treated for 4 h with WT or indicated mutant forms of PCSK9 (2.5 μg/ml). Biotinylated cell surface LDLRs were isolated and quantified by Western blotting using transferrin receptor (TfR) as a loading control. Shown are representative western blots ( top ) with densitometric analysis of three independent experiments ( bottom ). Error bars represent SEM ( n = 3). Significant change in LDLR expression compared to WT-PCSK9 was determined by Student’s t -test: ** p < 0.01. (C) Conditioned cell culture medium containing WT-PCSK9 (WT) or indicated mutants were incubated with LDL prior to density gradient-ultracentrifugation and immunoprecipitation and Western blot analysis of LDL-containing fractions. Shown is a representative experiment ( n = 3)

Article Snippet: We obtained fetal bovine serum (FBS) and newborn calf serum from ThermoFisher, EDTA-free Complete™ Protease Inhibitor Tablets were from Roche, Optiprep™ density gradient medium (60% w/v iodixanol) from Axis-Shield, NP-40 detergent was from Biovision and PolyJet DNA transfection reagent was from FroggaBio (Toronto, Ontario).

Techniques: Binding Assay, Transfection, Expressing, SDS Page, Western Blot, Cell Culture, Mutagenesis, Isolation, Incubation, Immunoprecipitation