Journal: PLoS ONE

Article Title: Common Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Epitopes Mediate Multiple Routes for Internalization and Function

doi: 10.1371/journal.pone.0125127

Figure Lengend Snippet: (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.

Article Snippet: Nitrocellulose membranes were blocked with Odyssey blocking buffer (Licor Biotechnologies), incubated with primary antibodies (LDLR (goat anti-Human; R&D Systems), TFNR (mouse anti-Human; Life Technologies), PCSK9 (sheep anti-Human; R&D Systems), APLP2 (goat anti-Human; R&D Systems), APP (goat anti-mouse; Life Technologies), APOER2 (Abcam)) for at least 1 hour, washed, and incubated with secondary antibodies (donkey anti-mouse 680, donkey anti-goat 800, goat anti-rabbit 680, goat anti-mouse 800 (Licor), or donkey anti-sheep 680 (Life Technologies)) before imaging on a Licor Odyssey imaging system.

Techniques:

Journal: PLoS ONE

Article Title: Common Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Epitopes Mediate Multiple Routes for Internalization and Function

doi: 10.1371/journal.pone.0125127

Figure Lengend Snippet: (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.

Article Snippet: Nitrocellulose membranes were blocked with Odyssey blocking buffer (Licor Biotechnologies), incubated with primary antibodies (LDLR (goat anti-Human; R&D Systems), TFNR (mouse anti-Human; Life Technologies), PCSK9 (sheep anti-Human; R&D Systems), APLP2 (goat anti-Human; R&D Systems), APP (goat anti-mouse; Life Technologies), APOER2 (Abcam)) for at least 1 hour, washed, and incubated with secondary antibodies (donkey anti-mouse 680, donkey anti-goat 800, goat anti-rabbit 680, goat anti-mouse 800 (Licor), or donkey anti-sheep 680 (Life Technologies)) before imaging on a Licor Odyssey imaging system.

Techniques: Western Blot, Immunoprecipitation, Control

Journal: PLoS ONE

Article Title: Common Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Epitopes Mediate Multiple Routes for Internalization and Function

doi: 10.1371/journal.pone.0125127

Figure Lengend Snippet: (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.

Article Snippet: Nitrocellulose membranes were blocked with Odyssey blocking buffer (Licor Biotechnologies), incubated with primary antibodies (LDLR (goat anti-Human; R&D Systems), TFNR (mouse anti-Human; Life Technologies), PCSK9 (sheep anti-Human; R&D Systems), APLP2 (goat anti-Human; R&D Systems), APP (goat anti-mouse; Life Technologies), APOER2 (Abcam)) for at least 1 hour, washed, and incubated with secondary antibodies (donkey anti-mouse 680, donkey anti-goat 800, goat anti-rabbit 680, goat anti-mouse 800 (Licor), or donkey anti-sheep 680 (Life Technologies)) before imaging on a Licor Odyssey imaging system.

Techniques: Western Blot, Immunoprecipitation, Recombinant

Journal: PLoS ONE

Article Title: Common Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Epitopes Mediate Multiple Routes for Internalization and Function

doi: 10.1371/journal.pone.0125127

Figure Lengend Snippet: (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.

Article Snippet: Nitrocellulose membranes were blocked with Odyssey blocking buffer (Licor Biotechnologies), incubated with primary antibodies (LDLR (goat anti-Human; R&D Systems), TFNR (mouse anti-Human; Life Technologies), PCSK9 (sheep anti-Human; R&D Systems), APLP2 (goat anti-Human; R&D Systems), APP (goat anti-mouse; Life Technologies), APOER2 (Abcam)) for at least 1 hour, washed, and incubated with secondary antibodies (donkey anti-mouse 680, donkey anti-goat 800, goat anti-rabbit 680, goat anti-mouse 800 (Licor), or donkey anti-sheep 680 (Life Technologies)) before imaging on a Licor Odyssey imaging system.

Techniques: Western Blot, Control, Binding Assay

Journal: PLoS ONE

Article Title: Common Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Epitopes Mediate Multiple Routes for Internalization and Function

doi: 10.1371/journal.pone.0125127

Figure Lengend Snippet: (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.

Article Snippet: APLP2-ECD was detected using mouse anti-human APLP2 and goat anti-Mouse HRP antibody (R&D Systems), followed by TMB (Thermo-Fisher) according to manufacturer’s instructions.

Techniques:

Journal: PLoS ONE

Article Title: Common Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Epitopes Mediate Multiple Routes for Internalization and Function

doi: 10.1371/journal.pone.0125127

Figure Lengend Snippet: (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.

Article Snippet: APLP2-ECD was detected using mouse anti-human APLP2 and goat anti-Mouse HRP antibody (R&D Systems), followed by TMB (Thermo-Fisher) according to manufacturer’s instructions.

Techniques: Western Blot, Immunoprecipitation, Control

Journal: PLoS ONE

Article Title: Common Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Epitopes Mediate Multiple Routes for Internalization and Function

doi: 10.1371/journal.pone.0125127

Figure Lengend Snippet: (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.

Article Snippet: APLP2-ECD was detected using mouse anti-human APLP2 and goat anti-Mouse HRP antibody (R&D Systems), followed by TMB (Thermo-Fisher) according to manufacturer’s instructions.

Techniques: Western Blot, Immunoprecipitation, Recombinant

Journal: PLoS ONE

Article Title: Common Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Epitopes Mediate Multiple Routes for Internalization and Function

doi: 10.1371/journal.pone.0125127

Figure Lengend Snippet: (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.

Article Snippet: APLP2-ECD was detected using mouse anti-human APLP2 and goat anti-Mouse HRP antibody (R&D Systems), followed by TMB (Thermo-Fisher) according to manufacturer’s instructions.

Techniques: Western Blot, Control, Binding Assay

Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: SNX17-Retriever/CCC/WASH pathway is essential for recycling of Reelin signaling receptors, APP family, and SLITRK family proteins. (A) Representative blots of co-expression of mCherry-tagged SNX17 with GFP-tagged cytoplasmic tail of LRP8 and VLDLR from three independent experiments. The interactions between mCherry-SNX17 and wild-type GFP-tagged cytoplasmic tails of Human-LRP8 and Human-VLDLR were significantly decreased when NPxY was substituted with NPxA. (B) Representative blots of analysis from three independent experiments using DIV17 rat cortical neurons transduced with either a scramble-control, SNX17, or VPS35L shRNA. Bar graphs show relative protein abundance of LRP8 and VLDLR in cell lysate or cell surface. (C) DIV17 rat cortical neurons transduced with shRNA were incubated for 30 min with or without AP-Reelin, followed by cell lysis and western blot analysis. Bar graph shows quantification of band intensities of AP relative to cells transduced with sh-SCR control from three independent experiments. (D) DIV17 rat cortical neurons transduced with shRNA were incubated for 7 min with or without Reelin. Phosphorylation level of Dab1 was then measured using immunoprecipitation and western blot analysis. Representative blots and quantification from three independent analyses are shown. (E) Volcano plots of transmembrane proteins with decreased (blue circles) or increased (red circles) cell surface abundance in sh-VPS35L suppression comparedto sh-SCR in DIV17 rat cortical neuron from three independent TMT-based proteomic experiments. Two independent sh-RNAs for VPS35L were used to avoid off-target effects. (F) Enrichment analysis of significantly downregulated proteins (LogFC < −0.32, p < 0.05) in the sh-VPS35L compared to sh-SCR by Metascape. (G) Representative blots of mCherry-nanotrap for mCherry-SNX17 under co-overexpression of chimeric constructs of Human-SLITRK family proteins from three independent experiments. All proteins except for SLITRK4 have an NPxY motif, which was mutated to NPxA in the mutant. (H) Representative blots of mCherry-nanotrap for mCherry-SNX17 under co-overexpression of chimeric constructs of Human-APP family proteins from three independent experiments. APP, APLP1, and APLP2 have NPxY motifs, and the NPxY motif was mutated to NPxA in the mutant. (I) Representative blots for APP and APLP2 in rat cortical neurons. Cell surface protein fraction was obtained, and N-Cadherin were used for loading control. Bar graphs show relative values of cells with sh-SNX17 or sh-VPS35L suppression compared to sh-SCR control cells (n=3). In all graphs, error bars represent mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: The following antibodies were used in this study (WB: western blot, IF: immunofluorescence): rabbit anti-SNX17 (Proteintech, 10275-1-AP, WB), mouse anti-GFP (Roche, 11814460001, WB), rabbit anti-GFP (GeneTex, GTX30738, WB), mouse anti-mCherry (antibodies.com, A85305, WB), rabbit anti-mCherry (antibodies.com, A85306, WB), rabbit anti-CCDC22, (Proteintech, 16636-1-AP, WB), mouse anti-CCDC93, (Origene, CF800568, WB), rabbit anti COMMD4 and rabbit anti COMMD9 (kind gift from Prof. Ezra Burstein, WB) rabbit anti-C16orf62 (Abcam, ab97889, WB), rabbit anti-C16orf62 (Pierce, PA5-28553, IF), rabbit anti-DSCR3 (Merck Millipore, ABN87, WB), rabbit anti-integrin-β1 (Abcam, ab52971, WB), goat anti-VPS35 (antibodies.com, A83699, IF), mouse anti-VPS29 (Santa Cruz, sc-398874, WB), rabbit anti-KIAA1033 (Proteintech, 51101-1-AP, WB), mouse anti-Strumpellin (Santa Cruz, sc-377146, WB), mouse anti-β actin (Sigma, A1978, WB), rabbit anti-LRP1 (Abcam, ab92544, WB), rabbit anti-LRP2 (Proteintech, 19700-1-AP, WB), rabbit anti-LRP2 (prepared as described in , IHC), rabbit anti-LRP4 (SIGMA, HPA012300, WB), rabbit anti-LRP8 (Abcam, ab108208, WB), mouse anti-VLDLR (Santa Cruz, sc-18824, WB), rabbit anti-APP (Abcam, ab32136, WB), rabbit anti-APLP2 (Proteintech, 15041-1-AP, WB), mouse anti-AP (Thermo Fisher, MA1-20245, WB), rabbit anti-Dab1 (kind gift from Dr. M Hattori, IP/WB) , mouse anti-Phosphotyrosine (Merck, 05-321, WB), rabbit anti-GLUT1 (Abcam, ab115730, IF/WB), mouse anti-N-cadherin (Cell signalling technology, 14215S, WB), anti-PSD95 (Merck, MAB1596, WB), mouse anti-FLAG (SIGMA, F1804, WB), rabbit anti-FGFR2, (Proteintech, 13042-1-AP, WB), rabbit anti-ERK1/2 (Cell Signaling Technology, 9102, WB), rabbit anti-phospho-ERK1/2 (Cell Signaling Technology, 9101, WB), anti Ctip2 (Abcam, ab18465, IHC), anti Tbr1 (Abcam, ab275960, IHC), anti Neun (Cell signalling technology, 12943, IHC).

Techniques: Expressing, Transduction, Control, shRNA, Incubation, Lysis, Western Blot, Immunoprecipitation, Over Expression, Construct, Mutagenesis