Determining which of APP or APLP2 affects PCSK9 internalization.

<p>(A, B, and C) PCSK9-488 internalization in the presence of mIC and hIC antibodies (top row), mIC and J16 (middle top row), hIC and 5F6 (middle bottom row), or 5F6 and J16 (bottom row) in (A) Neg, (B) APLP2, or (C) APP siRNA treated HepG2 cells. Internalized human and mouse antibodies shown in blue and red, respectively. Scale bars, 10 μM. (D) Quantification of (A, B, and C) shown as average fluorescent signal of PCSK9-488 per cell normalized to IC with SEM of 3 independent experiments.</p

APLP2 and LDLR interactions with PCSK9 and their regulation of PCSK9 function.

<p>(A and B) Western blot showing APLP2, PCSK9, or Transferrin receptor (TFNR) levels in input fraction (I), IC or J16 immunoprecipitated samples (IP Ab.) in the absence or presence of 5F6 Fab or 12E3 Fab, as indicated. (B) Quantification of (A); shown as average APLP2 normalized to PCSK9 of 3 independent experiments with SEM. (C and D) J16 coIPs of PCSK9 from Neg or LDLR siRNA treated HepG2 cells with IC control, as indicated. (D) Quantification of (C); shown as average APLP2 normalized to PCSK9 from 3 independent experiments with SEM. (E, F, and G) Western blot of LDLR, APOER2, or TFNR in siRNA treated cells following treatment with PCSK9 at 0, 20, 50, or 100 μg/ml. (F) LDLR levels from (E) quantified as percent LDLR degradation of untreated cells and normalized to Neg siRNA samples. Shown as average with SEM from 4 independent experiments. (G) Same as F, but measuring APOER2 levels.</p

PCSK9-488 internalization and LDLR levels <i>in vivo</i>.

<p>PCSK9-488 (green) internalization in mouse liver in the presence of mIC and hIC (top row), mIC and J16 (middle top row), hIC and 5F6 (middle bottom row), or 5F6 and J16 (bottom row). LDLR (red) and DAPI (blue) staining shown, as indicated. Scale bars, 10 μM.</p

ApoB/LDL effects on PCSK9 internalization and function.

<p>(A and B) PCSK9-488 internalization in the presence of J16 (top row), LDL and J16 (middle row), or LDL+5F6+J16 (bottom row) in APLP2 siRNA treated cells. Dotted line indicates background signal, as measured by IC alone. Scale bars, 10 μM. (B) Quantification of (A) shown as average+SEM of J16 fluorescent signal per cell in APLP2 siRNA treated cells from 3 independent experiments. Dotted line indicates average IC background levels. Scale bars, 10 μM. (C) Representative western blot showing APLP2, LDLR, ApoB, Transferrin receptor (TFNR) levels in input fraction (I), IC or J16 immunoprecipitated samples (IP Ab.) under pH 7.4 or pH 6.0 conditions with increasing concentrations of ApoB, as indicated. (D) Western blot showing recombinant ApoB, LDLR-ECD, or PCSK9 in anti-LDLR immunoprecipitated samples at pH 6.0, with or without 5F6 Fab, as indicated. All experiments were performed independently at least 3 times and representative data are shown here.</p

PCSK9 follows both direct and indirect internalization routes.

<p>(A) Western blot of liver lysates taken from negative siRNA or APLP2 siRNA treated mice showing relative APLP2 levels as compared to Actin loading control. (B) Internalization of J16 bound PCSK9 in liver in negative or APLP2 siRNA treated mice. Scale bars, 10 μM. (C) Schematic depicting interactions of the proposed direct and indirect PCSK9 internalization routes. At the cell surface, PCSK9 can bind directly to LDLR or APLP2; PCSK9 binding to APLP2 requires LDLR/APLP2 interactions. For both direct routes, following endocytosis, PCSK9 bridges LDLR to APLP2, and APLP2 mediates lysosomal delivery of the complex. Indirect PCSK9 internalization is mediated via LDL. PCSK9 binds LDL, and LDL binds LDLR at the cell surface. Following endocytosis, PCSK9 can potentially bridge dissociated LDL to LDLR.</p