Precast mini gels

Animal studies were performed in accordance with the University of Pennsylvania, institutional review board (IRB). LDLR^-/-, APOBEC-1^-/- double knockout (DKO) and LDLR^-/-, APOBEC-1^-/-, human ApoB100 transgenic (LAHB) mice were injected via tail vein for vector administration and serum collected by retro-orbital bleeds. At end of study animals were sacrificed and livers harvested for analysis. Vector were obtained from the Vector Core at the University of Pennsylvania and expressed cDNAs for hLDLR, hPCSK9 or hIDOL driven from a thyroxine binding globulin promoter (TBG). Mutations were introduced into wild type hLDLR using the Quick Change lightning kit (Agilent). Serum cholesterol levels were analyzed on a MIRA analyzer (Roche). Western blotting was done using precast mini gels (Invitrogen) and probed using a polyclonal hLDLR antibody. In vitro LDLR assay was performed by transiently transfecting HEK293 cells and pulsing the cells the following day with BODIPY labeled LDL (Invitrogen). Data were analyzed using one-way Analysis of Variance models with pair-wise group differences in mean cholesterol level assessed using Tukey's post-hoc tests.

Cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5% Tween-20, 2% Igepal CA-630, 2 mM PMSF, 50 mM β-glycerophosphate (Merck) and protease inhibitor cocktail tablet (cOmplete Mini, Roche Diagnostics). Equal amounts of lysates were loaded into precast mini-gels (Invitrogen) and resolved by SDS–PAGE. Transfer of proteins onto nitrocellulose membranes and incubation with primary/secondary antibodies were performed according to standard procedures. Visualization of protein bands was achieved by fluorescence imaging on the Odyssey Clx system (LI-COR Biosciences). Antibodies and dilutions used were as follows: anti-WRN (1:5,000, Novus), anti-pKAP1 (Bethyl Laboratories, 1:100), anti-tubulin (1:5,000, sigma), anti-MLH1 (Cell Signaling Technology, 3515, 1:1,000), anti-MSH2 (Abcam, ab52266, 1:1,000), anti-GAPDH (Cell Signaling Technology, 5174, 1:5,000), IRDye 680RD Goat anti-Mouse IgG (1:2,000, LI-COR Biosciences), IRDye 800CW Goat anti-Rabbit IgG (1:2,000, LI-COR Biosciences), Goat anti-Rabbit Alexa Fluor 647 (ThermoFisher, A27040, 1:5,000).