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  • 86
    ABclonal Biotechnology antibody to twist a3237
    Antibody To Twist A3237, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher streptavidin
    CA projections C1a, e, and c are lost in the pf16 mutant, and the PF16 C-terminus locates to the C1a projection. (A – P) Tomographic slices (columns 1 and 2) and isosurface renderings (columns 3 and 4) of the averaged CA repeats of WT (A–D), 9+2 pf16 (E and F), and the tagged rescue pf16;PF16::BCCP without adding nanogold (I–L, control) and after treatment with <t>streptavidin</t> gold (M–P) in cross-sectional (columns 1 and 3) and longitudinal views (columns 2 and 4). The thin blue line in A indicates the location of the slices shown in column 2. The C1a, e, and c projections (indicated by black brackets in A and C) were missing in the pf16 mutant (white bracket in E). When the C-terminus of PF16 was tagged with BCCP, the additional density of the BCCP-streptavidin-gold label was detected in the C1a projection (blue arrowheads in M–P); this label density was not observed in WT (white arrowheads in A–D) or control samples (white arrowheads in I–L). Scale bar in N, 20 nm (valid for all EM images).
    Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore apoe4 proteins
    A. Schematic overview of 4 h treatment with 2.5 μg/ml recombinant ApoE3, <t>ApoE4</t> or vehicle control in N2a cells. B. Representative epifluorescence images of N2a cells treated with recombinant ApoE3 or ApoE4 for 4 h showing an overview of human ApoE internalization in N2a cells. The N2a cells were labeled for ApoE (green), DAPI (blue) and phalloidin (grey). Cells showing internalized human ApoE were indicated by arrows, cells negative for ApoE were indicated by arrowheads. Scale bar is 40 μm. C. Higher magnification image of ApoE3- and ApoE4-treated N2a cells from . Internalized recombinant ApoE3 and ApoE4 puncta detected in N2a cells show a vesicle-like pattern. Scale bar represents 40 μm. D. Representative images obtained by epifluorescence microscopy of N2a cells incubated with recombinant ApoE3 or ApoE4 for 4 h. The cells were labeled for ApoE (green), late endosomal/lysosomal marker LAMP1 (red), DAPI (blue) and phalloidin (grey). The scale bar represents 10 μm. E. Quantification of , showing the co-localization levels (in percentages) of ApoE with LAMP1. F. Representative epifluorescence images of recombinant ApoE3- and ApoE4-treated N2a cells labeled for ApoE (green), late endosomal marker Rab7 (red), DAPI (blue) and phalloidin (grey). Scale bar is equal to 10 μm. G. Quantification of ApoE-Rab7 co-localization of . H. Representative images taken by epifluorescence microscopy of N2a cells showing LAMP1 (red) overlaps with Filipin (grey), a free cholesterol dye. Scale bar is 20 μm. I. Schematic representation of astrocyte-conditioned media collection from ApoE KO, ApoE3 KI and ApoE4 KI primary astrocytes, followed by media incubation of N2a cells for 4 h. J. Representative western blot of secreted human ApoE proteins detected in astrocyte conditioned media after culturing 48 h with ApoE KO, ApoE3 KI and ApoE4 KI primary astrocytes. K-L. Representative orthogonal images obtained by confocal microscopy showing ApoE (green) co-localizing with LAMP1 (red) (K) and to a lower extent also with Rab7 (red) (L) in ApoE3 and ApoE4 astrocyte conditioned media treated N2a cells. The cells were further labeled for DAPI (blue) and phalloidin (grey). Arrows indicate ApoE puncta inside LAMP1- (K) and Rab7- (L) positive vesicles. Scale bars represent 5 μm. Data is expressed as mean ± SD. ns = non-significant, Rec ApoE = recombinant ApoE. See also Supplementary Figure 1A-C.
    Apoe4 Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apoe4 proteins/product/Millipore
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    86
    Thermo Fisher alexa488
    A. Schematic overview of 4 h treatment with 2.5 μg/ml recombinant ApoE3, <t>ApoE4</t> or vehicle control in N2a cells. B. Representative epifluorescence images of N2a cells treated with recombinant ApoE3 or ApoE4 for 4 h showing an overview of human ApoE internalization in N2a cells. The N2a cells were labeled for ApoE (green), DAPI (blue) and phalloidin (grey). Cells showing internalized human ApoE were indicated by arrows, cells negative for ApoE were indicated by arrowheads. Scale bar is 40 μm. C. Higher magnification image of ApoE3- and ApoE4-treated N2a cells from . Internalized recombinant ApoE3 and ApoE4 puncta detected in N2a cells show a vesicle-like pattern. Scale bar represents 40 μm. D. Representative images obtained by epifluorescence microscopy of N2a cells incubated with recombinant ApoE3 or ApoE4 for 4 h. The cells were labeled for ApoE (green), late endosomal/lysosomal marker LAMP1 (red), DAPI (blue) and phalloidin (grey). The scale bar represents 10 μm. E. Quantification of , showing the co-localization levels (in percentages) of ApoE with LAMP1. F. Representative epifluorescence images of recombinant ApoE3- and ApoE4-treated N2a cells labeled for ApoE (green), late endosomal marker Rab7 (red), DAPI (blue) and phalloidin (grey). Scale bar is equal to 10 μm. G. Quantification of ApoE-Rab7 co-localization of . H. Representative images taken by epifluorescence microscopy of N2a cells showing LAMP1 (red) overlaps with Filipin (grey), a free cholesterol dye. Scale bar is 20 μm. I. Schematic representation of astrocyte-conditioned media collection from ApoE KO, ApoE3 KI and ApoE4 KI primary astrocytes, followed by media incubation of N2a cells for 4 h. J. Representative western blot of secreted human ApoE proteins detected in astrocyte conditioned media after culturing 48 h with ApoE KO, ApoE3 KI and ApoE4 KI primary astrocytes. K-L. Representative orthogonal images obtained by confocal microscopy showing ApoE (green) co-localizing with LAMP1 (red) (K) and to a lower extent also with Rab7 (red) (L) in ApoE3 and ApoE4 astrocyte conditioned media treated N2a cells. The cells were further labeled for DAPI (blue) and phalloidin (grey). Arrows indicate ApoE puncta inside LAMP1- (K) and Rab7- (L) positive vesicles. Scale bars represent 5 μm. Data is expressed as mean ± SD. ns = non-significant, Rec ApoE = recombinant ApoE. See also Supplementary Figure 1A-C.
    Alexa488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa488/product/Thermo Fisher
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    86
    Millipore apoe4
    A. Schematic overview of 4 h treatment with 2.5 μg/ml recombinant ApoE3, <t>ApoE4</t> or vehicle control in N2a cells. B. Representative epifluorescence images of N2a cells treated with recombinant ApoE3 or ApoE4 for 4 h showing an overview of human ApoE internalization in N2a cells. The N2a cells were labeled for ApoE (green), DAPI (blue) and phalloidin (grey). Cells showing internalized human ApoE were indicated by arrows, cells negative for ApoE were indicated by arrowheads. Scale bar is 40 μm. C. Higher magnification image of ApoE3- and ApoE4-treated N2a cells from . Internalized recombinant ApoE3 and ApoE4 puncta detected in N2a cells show a vesicle-like pattern. Scale bar represents 40 μm. D. Representative images obtained by epifluorescence microscopy of N2a cells incubated with recombinant ApoE3 or ApoE4 for 4 h. The cells were labeled for ApoE (green), late endosomal/lysosomal marker LAMP1 (red), DAPI (blue) and phalloidin (grey). The scale bar represents 10 μm. E. Quantification of , showing the co-localization levels (in percentages) of ApoE with LAMP1. F. Representative epifluorescence images of recombinant ApoE3- and ApoE4-treated N2a cells labeled for ApoE (green), late endosomal marker Rab7 (red), DAPI (blue) and phalloidin (grey). Scale bar is equal to 10 μm. G. Quantification of ApoE-Rab7 co-localization of . H. Representative images taken by epifluorescence microscopy of N2a cells showing LAMP1 (red) overlaps with Filipin (grey), a free cholesterol dye. Scale bar is 20 μm. I. Schematic representation of astrocyte-conditioned media collection from ApoE KO, ApoE3 KI and ApoE4 KI primary astrocytes, followed by media incubation of N2a cells for 4 h. J. Representative western blot of secreted human ApoE proteins detected in astrocyte conditioned media after culturing 48 h with ApoE KO, ApoE3 KI and ApoE4 KI primary astrocytes. K-L. Representative orthogonal images obtained by confocal microscopy showing ApoE (green) co-localizing with LAMP1 (red) (K) and to a lower extent also with Rab7 (red) (L) in ApoE3 and ApoE4 astrocyte conditioned media treated N2a cells. The cells were further labeled for DAPI (blue) and phalloidin (grey). Arrows indicate ApoE puncta inside LAMP1- (K) and Rab7- (L) positive vesicles. Scale bars represent 5 μm. Data is expressed as mean ± SD. ns = non-significant, Rec ApoE = recombinant ApoE. See also Supplementary Figure 1A-C.
    Apoe4, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apoe4/product/Millipore
    Average 86 stars, based on 1 article reviews
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    Image Search Results


    CA projections C1a, e, and c are lost in the pf16 mutant, and the PF16 C-terminus locates to the C1a projection. (A – P) Tomographic slices (columns 1 and 2) and isosurface renderings (columns 3 and 4) of the averaged CA repeats of WT (A–D), 9+2 pf16 (E and F), and the tagged rescue pf16;PF16::BCCP without adding nanogold (I–L, control) and after treatment with streptavidin gold (M–P) in cross-sectional (columns 1 and 3) and longitudinal views (columns 2 and 4). The thin blue line in A indicates the location of the slices shown in column 2. The C1a, e, and c projections (indicated by black brackets in A and C) were missing in the pf16 mutant (white bracket in E). When the C-terminus of PF16 was tagged with BCCP, the additional density of the BCCP-streptavidin-gold label was detected in the C1a projection (blue arrowheads in M–P); this label density was not observed in WT (white arrowheads in A–D) or control samples (white arrowheads in I–L). Scale bar in N, 20 nm (valid for all EM images).

    Journal: The Journal of Cell Biology

    Article Title: Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

    doi: 10.1083/jcb.201906006

    Figure Lengend Snippet: CA projections C1a, e, and c are lost in the pf16 mutant, and the PF16 C-terminus locates to the C1a projection. (A – P) Tomographic slices (columns 1 and 2) and isosurface renderings (columns 3 and 4) of the averaged CA repeats of WT (A–D), 9+2 pf16 (E and F), and the tagged rescue pf16;PF16::BCCP without adding nanogold (I–L, control) and after treatment with streptavidin gold (M–P) in cross-sectional (columns 1 and 3) and longitudinal views (columns 2 and 4). The thin blue line in A indicates the location of the slices shown in column 2. The C1a, e, and c projections (indicated by black brackets in A and C) were missing in the pf16 mutant (white bracket in E). When the C-terminus of PF16 was tagged with BCCP, the additional density of the BCCP-streptavidin-gold label was detected in the C1a projection (blue arrowheads in M–P); this label density was not observed in WT (white arrowheads in A–D) or control samples (white arrowheads in I–L). Scale bar in N, 20 nm (valid for all EM images).

    Article Snippet: On the next day, the slides were washed four times over 1 h with blocking buffer, and then treated for 1 h with blocking buffer containing the secondary antibody (F(ab′)2-goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 568, Invitrogen, A11019, 1:1,000) and Streptavidin (Alexa Fluor 488, Invitrogen, A32354, 1:200).

    Techniques: Mutagenesis

    The FAP76 N-terminus is located at the interface between C1c and C1e projections. (A) Immunofluorescence light microscopy images of axonemes isolated from fap76-1 (left) and fap76-1;BCCP::FAP76 rescue (right) probed by anti-acetylated-tubulin antibody (red) and fluorescently tagged streptavidin (green). The streptavidin signal indicates that the BCCP-tagged FAP76 was assembled into the axoneme of the rescued strain. (B) SDS–polyacrylamide gel stained by a silver enhancement kit (top) to show that a specific band of appropriate relative mobility could be detected in the fap76-1;BCCP::FAP76 axonemes treated with streptavidin-Au, but not in the control (–Au). Coomassie brilliant blue (CBB) staining (bottom) shows the tubulin bands as loading controls. (C) A classification analysis of the C1c psu1 (black arrows in E and F) in WT and fap76-1 , and fap76-1;BCCP::FAP76 rescue axonemes (white arrows in H, I, K, and L). The particle numbers (n) included in the averages for WT, fap76-1 , fap76-1;BCCP::FAP76 (+Au), and fap76-1;BCCP::FAP76 (–Au) were 664, 927, 1,089, and 935 (see Table S3). (D–L) Comparison of tomographic slices (D, E, G, H, J, and K) and isosurface renderings (F, I, and L) of the averaged CA repeats of WT (D–F) versus fap76-1;BCCP::FAP76 rescue strain either without (G–I) or treated with (J–L) streptavidin gold, viewed in cross-sectional (D, G, and J) and longitudinal (E, F, H, I, K, and L) orientations. The additional density of the streptavidin-gold label at the interface between the C1c and C1e projections in the gold-treated rescue strain (blue arrowheads in J–L) is not observed in WT or control CA (white arrowheads in D–I). Thin blue line in D indicates the location for the tomographic slices shown in E, H, and K. Scale bar in A, 5 µm (valid for all fluorescence images); K, 20 nm (valid for all EM images in D–L).

    Journal: The Journal of Cell Biology

    Article Title: Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

    doi: 10.1083/jcb.201906006

    Figure Lengend Snippet: The FAP76 N-terminus is located at the interface between C1c and C1e projections. (A) Immunofluorescence light microscopy images of axonemes isolated from fap76-1 (left) and fap76-1;BCCP::FAP76 rescue (right) probed by anti-acetylated-tubulin antibody (red) and fluorescently tagged streptavidin (green). The streptavidin signal indicates that the BCCP-tagged FAP76 was assembled into the axoneme of the rescued strain. (B) SDS–polyacrylamide gel stained by a silver enhancement kit (top) to show that a specific band of appropriate relative mobility could be detected in the fap76-1;BCCP::FAP76 axonemes treated with streptavidin-Au, but not in the control (–Au). Coomassie brilliant blue (CBB) staining (bottom) shows the tubulin bands as loading controls. (C) A classification analysis of the C1c psu1 (black arrows in E and F) in WT and fap76-1 , and fap76-1;BCCP::FAP76 rescue axonemes (white arrows in H, I, K, and L). The particle numbers (n) included in the averages for WT, fap76-1 , fap76-1;BCCP::FAP76 (+Au), and fap76-1;BCCP::FAP76 (–Au) were 664, 927, 1,089, and 935 (see Table S3). (D–L) Comparison of tomographic slices (D, E, G, H, J, and K) and isosurface renderings (F, I, and L) of the averaged CA repeats of WT (D–F) versus fap76-1;BCCP::FAP76 rescue strain either without (G–I) or treated with (J–L) streptavidin gold, viewed in cross-sectional (D, G, and J) and longitudinal (E, F, H, I, K, and L) orientations. The additional density of the streptavidin-gold label at the interface between the C1c and C1e projections in the gold-treated rescue strain (blue arrowheads in J–L) is not observed in WT or control CA (white arrowheads in D–I). Thin blue line in D indicates the location for the tomographic slices shown in E, H, and K. Scale bar in A, 5 µm (valid for all fluorescence images); K, 20 nm (valid for all EM images in D–L).

    Article Snippet: On the next day, the slides were washed four times over 1 h with blocking buffer, and then treated for 1 h with blocking buffer containing the secondary antibody (F(ab′)2-goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 568, Invitrogen, A11019, 1:1,000) and Streptavidin (Alexa Fluor 488, Invitrogen, A32354, 1:200).

    Techniques: Immunofluorescence, Light Microscopy, Isolation, Staining, Fluorescence

    A. Schematic overview of 4 h treatment with 2.5 μg/ml recombinant ApoE3, ApoE4 or vehicle control in N2a cells. B. Representative epifluorescence images of N2a cells treated with recombinant ApoE3 or ApoE4 for 4 h showing an overview of human ApoE internalization in N2a cells. The N2a cells were labeled for ApoE (green), DAPI (blue) and phalloidin (grey). Cells showing internalized human ApoE were indicated by arrows, cells negative for ApoE were indicated by arrowheads. Scale bar is 40 μm. C. Higher magnification image of ApoE3- and ApoE4-treated N2a cells from . Internalized recombinant ApoE3 and ApoE4 puncta detected in N2a cells show a vesicle-like pattern. Scale bar represents 40 μm. D. Representative images obtained by epifluorescence microscopy of N2a cells incubated with recombinant ApoE3 or ApoE4 for 4 h. The cells were labeled for ApoE (green), late endosomal/lysosomal marker LAMP1 (red), DAPI (blue) and phalloidin (grey). The scale bar represents 10 μm. E. Quantification of , showing the co-localization levels (in percentages) of ApoE with LAMP1. F. Representative epifluorescence images of recombinant ApoE3- and ApoE4-treated N2a cells labeled for ApoE (green), late endosomal marker Rab7 (red), DAPI (blue) and phalloidin (grey). Scale bar is equal to 10 μm. G. Quantification of ApoE-Rab7 co-localization of . H. Representative images taken by epifluorescence microscopy of N2a cells showing LAMP1 (red) overlaps with Filipin (grey), a free cholesterol dye. Scale bar is 20 μm. I. Schematic representation of astrocyte-conditioned media collection from ApoE KO, ApoE3 KI and ApoE4 KI primary astrocytes, followed by media incubation of N2a cells for 4 h. J. Representative western blot of secreted human ApoE proteins detected in astrocyte conditioned media after culturing 48 h with ApoE KO, ApoE3 KI and ApoE4 KI primary astrocytes. K-L. Representative orthogonal images obtained by confocal microscopy showing ApoE (green) co-localizing with LAMP1 (red) (K) and to a lower extent also with Rab7 (red) (L) in ApoE3 and ApoE4 astrocyte conditioned media treated N2a cells. The cells were further labeled for DAPI (blue) and phalloidin (grey). Arrows indicate ApoE puncta inside LAMP1- (K) and Rab7- (L) positive vesicles. Scale bars represent 5 μm. Data is expressed as mean ± SD. ns = non-significant, Rec ApoE = recombinant ApoE. See also Supplementary Figure 1A-C.

    Journal: bioRxiv

    Article Title: Apolipoprotein E intersects with amyloid-β within neurons

    doi: 10.1101/2022.12.13.520238

    Figure Lengend Snippet: A. Schematic overview of 4 h treatment with 2.5 μg/ml recombinant ApoE3, ApoE4 or vehicle control in N2a cells. B. Representative epifluorescence images of N2a cells treated with recombinant ApoE3 or ApoE4 for 4 h showing an overview of human ApoE internalization in N2a cells. The N2a cells were labeled for ApoE (green), DAPI (blue) and phalloidin (grey). Cells showing internalized human ApoE were indicated by arrows, cells negative for ApoE were indicated by arrowheads. Scale bar is 40 μm. C. Higher magnification image of ApoE3- and ApoE4-treated N2a cells from . Internalized recombinant ApoE3 and ApoE4 puncta detected in N2a cells show a vesicle-like pattern. Scale bar represents 40 μm. D. Representative images obtained by epifluorescence microscopy of N2a cells incubated with recombinant ApoE3 or ApoE4 for 4 h. The cells were labeled for ApoE (green), late endosomal/lysosomal marker LAMP1 (red), DAPI (blue) and phalloidin (grey). The scale bar represents 10 μm. E. Quantification of , showing the co-localization levels (in percentages) of ApoE with LAMP1. F. Representative epifluorescence images of recombinant ApoE3- and ApoE4-treated N2a cells labeled for ApoE (green), late endosomal marker Rab7 (red), DAPI (blue) and phalloidin (grey). Scale bar is equal to 10 μm. G. Quantification of ApoE-Rab7 co-localization of . H. Representative images taken by epifluorescence microscopy of N2a cells showing LAMP1 (red) overlaps with Filipin (grey), a free cholesterol dye. Scale bar is 20 μm. I. Schematic representation of astrocyte-conditioned media collection from ApoE KO, ApoE3 KI and ApoE4 KI primary astrocytes, followed by media incubation of N2a cells for 4 h. J. Representative western blot of secreted human ApoE proteins detected in astrocyte conditioned media after culturing 48 h with ApoE KO, ApoE3 KI and ApoE4 KI primary astrocytes. K-L. Representative orthogonal images obtained by confocal microscopy showing ApoE (green) co-localizing with LAMP1 (red) (K) and to a lower extent also with Rab7 (red) (L) in ApoE3 and ApoE4 astrocyte conditioned media treated N2a cells. The cells were further labeled for DAPI (blue) and phalloidin (grey). Arrows indicate ApoE puncta inside LAMP1- (K) and Rab7- (L) positive vesicles. Scale bars represent 5 μm. Data is expressed as mean ± SD. ns = non-significant, Rec ApoE = recombinant ApoE. See also Supplementary Figure 1A-C.

    Article Snippet: Recombinant ApoE3 and ApoE4 proteins (Sigma-Aldrich, SRP4696 and A3234, respectively) were reconstituted in 0.1% bovine serum albumin (BSA) in milli-Q water to a stock concentration of 0.1 mg/ml.

    Techniques: Recombinant, Labeling, Epifluorescence Microscopy, Incubation, Marker, Western Blot, Confocal Microscopy

    A. Schematic overview of ApoE KO, ApoE3 KI and ApoE4 KI astrocyte conditioned medium collection and subsequent 4 h treatment to ApoE KO primary neuron cultures (19 DIV). B-C. Representative fluorescent images obtained by epifluorescence microscopy of ApoE KO neurons treated with ApoE3 or ApoE4 astrocyte conditioned medium for 4 h with the focus on neuronal cell bodies. The ApoE astrocyte medium-treated neurons were labeled for ApoE (green), neuronal dendrite marker MAP2 (grey) and either LAMP1 (B) or Rab7 (C) (both shown in magenta). The left panels (B-C) show an overview of the entire neuron; higher magnification images are shown in the other four panels. The white arrows indicate ApoE puncta overlap with LAMP1-positive vesicles in neuronal cell bodies (B) . Left panels: scale bars are 40 μm. Right panels: scale bars are 10 μm. D-G. Representative epifluorescence images of neurites from ApoE3 and ApoE4 media-treated primary neurons. The neurites are labeled with ApoE (green), MAP2 (D-E) (grey), and late endosomal/lysosomal marker LAMP1 (D) , late endosomal marker Rab7 (E) , autophagosomal marker LC3β (F) or early endosomal marker EEA1 (G) (all in magenta). White arrows indicate co-localization between ApoE and the subcellular markers (D-G) . Scale bars represent 10 μm. H-I. Representative fluorescence images of ApoE KO neurites treated with control (DMSO) or 10 nM lysosomal inhibitor bafilomycin A1 for 1 h, followed by 4 h ApoE3 ( H ) or ApoE4 ( I ) astrocyte conditioned media. The neurites were labeled for ApoE (green), LAMP1 (red) and MAP2 (grey). ApoE and LAMP1 co-localization, indicating the presence of ApoE at late endosomes and/or lysosomes, is indicated by white arrows. Scale bar is 15 μm. J-K. Quantification of ApoE and LAMP1 co-localization in ApoE3-treated neurons shown in ( J ) and ApoE4-treated neurons shown in ( K ) with and without bafilomycin A1 treatment. The researcher performing the quantifications was blinded. Data is shown as mean ± SD. See also Supplementary Figure 1D-E.

    Journal: bioRxiv

    Article Title: Apolipoprotein E intersects with amyloid-β within neurons

    doi: 10.1101/2022.12.13.520238

    Figure Lengend Snippet: A. Schematic overview of ApoE KO, ApoE3 KI and ApoE4 KI astrocyte conditioned medium collection and subsequent 4 h treatment to ApoE KO primary neuron cultures (19 DIV). B-C. Representative fluorescent images obtained by epifluorescence microscopy of ApoE KO neurons treated with ApoE3 or ApoE4 astrocyte conditioned medium for 4 h with the focus on neuronal cell bodies. The ApoE astrocyte medium-treated neurons were labeled for ApoE (green), neuronal dendrite marker MAP2 (grey) and either LAMP1 (B) or Rab7 (C) (both shown in magenta). The left panels (B-C) show an overview of the entire neuron; higher magnification images are shown in the other four panels. The white arrows indicate ApoE puncta overlap with LAMP1-positive vesicles in neuronal cell bodies (B) . Left panels: scale bars are 40 μm. Right panels: scale bars are 10 μm. D-G. Representative epifluorescence images of neurites from ApoE3 and ApoE4 media-treated primary neurons. The neurites are labeled with ApoE (green), MAP2 (D-E) (grey), and late endosomal/lysosomal marker LAMP1 (D) , late endosomal marker Rab7 (E) , autophagosomal marker LC3β (F) or early endosomal marker EEA1 (G) (all in magenta). White arrows indicate co-localization between ApoE and the subcellular markers (D-G) . Scale bars represent 10 μm. H-I. Representative fluorescence images of ApoE KO neurites treated with control (DMSO) or 10 nM lysosomal inhibitor bafilomycin A1 for 1 h, followed by 4 h ApoE3 ( H ) or ApoE4 ( I ) astrocyte conditioned media. The neurites were labeled for ApoE (green), LAMP1 (red) and MAP2 (grey). ApoE and LAMP1 co-localization, indicating the presence of ApoE at late endosomes and/or lysosomes, is indicated by white arrows. Scale bar is 15 μm. J-K. Quantification of ApoE and LAMP1 co-localization in ApoE3-treated neurons shown in ( J ) and ApoE4-treated neurons shown in ( K ) with and without bafilomycin A1 treatment. The researcher performing the quantifications was blinded. Data is shown as mean ± SD. See also Supplementary Figure 1D-E.

    Article Snippet: Recombinant ApoE3 and ApoE4 proteins (Sigma-Aldrich, SRP4696 and A3234, respectively) were reconstituted in 0.1% bovine serum albumin (BSA) in milli-Q water to a stock concentration of 0.1 mg/ml.

    Techniques: Epifluorescence Microscopy, Labeling, Marker, Fluorescence

    A. Representative fluorescence images of LAMP1-positive puncta (magenta) and ApoE (green) overlap in MAP2-negative cells in primary cultures. The primary brain cultures were treated with ApoE3 or ApoE4 astrocyte conditioned media for 4 h. The overlap of LAMP1 with human ApoE was highlighted by white arrows. Scale bars are 10 μm (bigger panels) and 3 μm (smaller panels). B. Representative images of wild-type, ApoE KO, ApoE3 KI and ApoE4 KI primary cultures labeled for neuronal marker MAP2 (magenta), astrocyte marker GFAP (green) and nuclear marker DAPI (white). Scale bar is 50 μm. C. Quantifications of MAP2-positive and GFAP-positive cells (%) in the primary cultures described in . The total number of cells was set based on the number of DAPI-positive nuclei present. Data is shown as mean ± SD. D. Representative fluorescence images of S100β-positive astrocytes (magenta) labeled for ApoE (green). The primary cultures containing the S100β-positive astrocytes were treated with ApoE3 or ApoE4 astrocyte conditioned media for 4 h. Scale bar represents 20 μm. WT = wild-type, PN = primary neurons.

    Journal: bioRxiv

    Article Title: Apolipoprotein E intersects with amyloid-β within neurons

    doi: 10.1101/2022.12.13.520238

    Figure Lengend Snippet: A. Representative fluorescence images of LAMP1-positive puncta (magenta) and ApoE (green) overlap in MAP2-negative cells in primary cultures. The primary brain cultures were treated with ApoE3 or ApoE4 astrocyte conditioned media for 4 h. The overlap of LAMP1 with human ApoE was highlighted by white arrows. Scale bars are 10 μm (bigger panels) and 3 μm (smaller panels). B. Representative images of wild-type, ApoE KO, ApoE3 KI and ApoE4 KI primary cultures labeled for neuronal marker MAP2 (magenta), astrocyte marker GFAP (green) and nuclear marker DAPI (white). Scale bar is 50 μm. C. Quantifications of MAP2-positive and GFAP-positive cells (%) in the primary cultures described in . The total number of cells was set based on the number of DAPI-positive nuclei present. Data is shown as mean ± SD. D. Representative fluorescence images of S100β-positive astrocytes (magenta) labeled for ApoE (green). The primary cultures containing the S100β-positive astrocytes were treated with ApoE3 or ApoE4 astrocyte conditioned media for 4 h. Scale bar represents 20 μm. WT = wild-type, PN = primary neurons.

    Article Snippet: Recombinant ApoE3 and ApoE4 proteins (Sigma-Aldrich, SRP4696 and A3234, respectively) were reconstituted in 0.1% bovine serum albumin (BSA) in milli-Q water to a stock concentration of 0.1 mg/ml.

    Techniques: Fluorescence, Labeling, Marker

    A. Schematic representation of experimental approach used. Astrocyte conditioned medium was collected from ApoE KO, ApoE3 KI and ApoE4 KI primary astrocytes and subsequently added to N2a APP Swe cells for 4 h. B. Representative orthogonal images obtained by confocal microscopy of N2a APP Swe cells incubated with ApoE3 and ApoE4 astrocyte conditioned media for 4 h. The cells were labeled for ApoE (green), human Aβ/APP-βCTFs by antibody 82e1 (red), DAPI (blue) and phalloidin (grey). Human ApoE and 82e1-positive Aβ/APP-βCTFs overlap as indicated by white arrows. C. Orthogonal fluorescent images of astrocytic ApoE3 and ApoE4-treated N2a APP Swe cells. ApoE was labeled in green, late endosomes/lysosomes with LAMP1 in red, DAPI in blue and phalloidin in grey. Arrows indicate overlap between ApoE- and LAMP1-positive puncta. D. Representative orthogonal confocal images of N2a APP Swe cells treated with astrocyte-derived ApoE3 and ApoE4 for 4 h. The cells are labeled for LAMP1-positive vesicles (green), 82e1-positive Aβ/APP-βCTFs (red) and DAPI (blue) to study the localization of Aβ-containing APP processing products in N2a APP Swe cells. White arrows highlights 82e1-positive puncta co-localizing with LAMP1-positive puncta. Scale bars are 5 μm ( B-D ). E. Schematic overview of ApoE KO, ApoE3 KI and ApoE4 KI astrocyte conditioned media collection and 4 h treatment of wild-type and AD APP/PS1 transgenic primary neurons (19 DIV). F. Representative confocal images of APP/PS1 neurons incubated with ApoE KO, ApoE3 or ApoE4 astrocyte conditioned media for 4 h. The intracellular intersection between internalized ApoE and endogenous human APP cleavage products was studied by labeling neurites for ApoE (green), APP metabolites Aβ/APP-βCTFs (82e1) (magenta) and MAP2 (grey). Arrows indicate ApoE and 82e1 co-localization at neurites. Scale bar is 15 μm. G. Higher magnification images of (indicated by a white box) of ApoE3 and ApoE4 astrocyte conditioned medium treated APP/PS1 neurons. The white arrows point at subcellular co-localization of internalized astrocyte-derived ApoE with endogenous human Aβ/APP-βCTFs. Scale bar is 6 μm. H. Schematic representation of synthetic 0.5 μM human Aβ 42 and astrocyte-derived ApoE3 or ApoE4 treatment in neurons that do not endogenously produce ApoE (ApoE KO). I. Representative confocal microscopy images of Aβ 42 and astrocytic ApoE-double treated primary neurons. The neurons were labeled for ApoE, APP/Aβ (6E10) and MAP2. Scale bar represents 20 μm. J. High magnification images of Aβ 42 and ApoE-treated neurons of the area shown by a white box in . White arrows indicate ApoE and APP/Aβ overlap; the white circle highlights overlap of larger puncta of ApoE and APP/Aβ. Scale bar is equal to 6 μm. See also Supplementary Figure 5A-B.

    Journal: bioRxiv

    Article Title: Apolipoprotein E intersects with amyloid-β within neurons

    doi: 10.1101/2022.12.13.520238

    Figure Lengend Snippet: A. Schematic representation of experimental approach used. Astrocyte conditioned medium was collected from ApoE KO, ApoE3 KI and ApoE4 KI primary astrocytes and subsequently added to N2a APP Swe cells for 4 h. B. Representative orthogonal images obtained by confocal microscopy of N2a APP Swe cells incubated with ApoE3 and ApoE4 astrocyte conditioned media for 4 h. The cells were labeled for ApoE (green), human Aβ/APP-βCTFs by antibody 82e1 (red), DAPI (blue) and phalloidin (grey). Human ApoE and 82e1-positive Aβ/APP-βCTFs overlap as indicated by white arrows. C. Orthogonal fluorescent images of astrocytic ApoE3 and ApoE4-treated N2a APP Swe cells. ApoE was labeled in green, late endosomes/lysosomes with LAMP1 in red, DAPI in blue and phalloidin in grey. Arrows indicate overlap between ApoE- and LAMP1-positive puncta. D. Representative orthogonal confocal images of N2a APP Swe cells treated with astrocyte-derived ApoE3 and ApoE4 for 4 h. The cells are labeled for LAMP1-positive vesicles (green), 82e1-positive Aβ/APP-βCTFs (red) and DAPI (blue) to study the localization of Aβ-containing APP processing products in N2a APP Swe cells. White arrows highlights 82e1-positive puncta co-localizing with LAMP1-positive puncta. Scale bars are 5 μm ( B-D ). E. Schematic overview of ApoE KO, ApoE3 KI and ApoE4 KI astrocyte conditioned media collection and 4 h treatment of wild-type and AD APP/PS1 transgenic primary neurons (19 DIV). F. Representative confocal images of APP/PS1 neurons incubated with ApoE KO, ApoE3 or ApoE4 astrocyte conditioned media for 4 h. The intracellular intersection between internalized ApoE and endogenous human APP cleavage products was studied by labeling neurites for ApoE (green), APP metabolites Aβ/APP-βCTFs (82e1) (magenta) and MAP2 (grey). Arrows indicate ApoE and 82e1 co-localization at neurites. Scale bar is 15 μm. G. Higher magnification images of (indicated by a white box) of ApoE3 and ApoE4 astrocyte conditioned medium treated APP/PS1 neurons. The white arrows point at subcellular co-localization of internalized astrocyte-derived ApoE with endogenous human Aβ/APP-βCTFs. Scale bar is 6 μm. H. Schematic representation of synthetic 0.5 μM human Aβ 42 and astrocyte-derived ApoE3 or ApoE4 treatment in neurons that do not endogenously produce ApoE (ApoE KO). I. Representative confocal microscopy images of Aβ 42 and astrocytic ApoE-double treated primary neurons. The neurons were labeled for ApoE, APP/Aβ (6E10) and MAP2. Scale bar represents 20 μm. J. High magnification images of Aβ 42 and ApoE-treated neurons of the area shown by a white box in . White arrows indicate ApoE and APP/Aβ overlap; the white circle highlights overlap of larger puncta of ApoE and APP/Aβ. Scale bar is equal to 6 μm. See also Supplementary Figure 5A-B.

    Article Snippet: Recombinant ApoE3 and ApoE4 proteins (Sigma-Aldrich, SRP4696 and A3234, respectively) were reconstituted in 0.1% bovine serum albumin (BSA) in milli-Q water to a stock concentration of 0.1 mg/ml.

    Techniques: Confocal Microscopy, Incubation, Labeling, Derivative Assay, Transgenic Assay

    A. Representative western blot bands of lysate of N2a APP Swe cells treated with ApoE KO, ApoE3 or ApoE4 astrocyte conditioned media or control (fresh N2a media)for 4 h (blue) and 8 h (orange). The western blot membranes were stained for APP, detected by 6E10 antibody, β-Actin and Aβ, detected using antibody 82e1. B-E. Quantification of western blots shown in . Full-length APP and Aβ protein levels were quantified in lysate of N2a APP Swe cells treated with astrocyte conditioned media for 4 h ( B and C , respectively, blue) or 8 h ( D and E, respectively, orange). Values were normalized to β-actin and control N2a APP Swe cells (APP and Aβ protein levels in these cells were set to 1). Data is shown as mean ± SD. F. Representative western blot membranes of sAPPα and Aβ protein levels in media from N2a APP Swe cells treated with ApoE KO, ApoE3 or ApoE4 astrocyte media or control (fresh N2a media) for 4 h (blue) or 8 h (orange). G-J. Quantification of secreted sAPPα and Aβ protein levels in the conditions shown in . sAPPα and Aβ levels were determined in N2a APP Swe cells treated with astrocyte media for 4 h ( G and H, respectively, blue) or 8 h ( I and J, respectively, orange). Data is shown as mean ± SD. * p-value < 0.05, ** p-value < 0.01. See also Supplementary Figure 6.

    Journal: bioRxiv

    Article Title: Apolipoprotein E intersects with amyloid-β within neurons

    doi: 10.1101/2022.12.13.520238

    Figure Lengend Snippet: A. Representative western blot bands of lysate of N2a APP Swe cells treated with ApoE KO, ApoE3 or ApoE4 astrocyte conditioned media or control (fresh N2a media)for 4 h (blue) and 8 h (orange). The western blot membranes were stained for APP, detected by 6E10 antibody, β-Actin and Aβ, detected using antibody 82e1. B-E. Quantification of western blots shown in . Full-length APP and Aβ protein levels were quantified in lysate of N2a APP Swe cells treated with astrocyte conditioned media for 4 h ( B and C , respectively, blue) or 8 h ( D and E, respectively, orange). Values were normalized to β-actin and control N2a APP Swe cells (APP and Aβ protein levels in these cells were set to 1). Data is shown as mean ± SD. F. Representative western blot membranes of sAPPα and Aβ protein levels in media from N2a APP Swe cells treated with ApoE KO, ApoE3 or ApoE4 astrocyte media or control (fresh N2a media) for 4 h (blue) or 8 h (orange). G-J. Quantification of secreted sAPPα and Aβ protein levels in the conditions shown in . sAPPα and Aβ levels were determined in N2a APP Swe cells treated with astrocyte media for 4 h ( G and H, respectively, blue) or 8 h ( I and J, respectively, orange). Data is shown as mean ± SD. * p-value < 0.05, ** p-value < 0.01. See also Supplementary Figure 6.

    Article Snippet: Recombinant ApoE3 and ApoE4 proteins (Sigma-Aldrich, SRP4696 and A3234, respectively) were reconstituted in 0.1% bovine serum albumin (BSA) in milli-Q water to a stock concentration of 0.1 mg/ml.

    Techniques: Western Blot, Staining

    A. Schematic visualization of the different primary neuron models used to study the effects of human ApoE isoforms on levels of intraneuronal Aβ 42 . B. Representative epifluorescence images of neuronal cell bodies from ApoE KO, ApoE3-KI and ApoE4-KI primary neurons (19 DIV). The neurons were labeled for endogenous mouse Aβ 42 using antibody 12F4 (16 colors) and MAP2 (magenta). Scale bar is 15 μm. C. Quantification of endogenous Aβ 42 in neuronal cell bodies as shown in . The levels of Aβ 42 were significantly higher in ApoE4-KI neurons compared to ApoE KO neurons. D. Representative images of ApoE KO, ApoE3-KI and ApoE4-KI neurites labeled for Aβ 42 by antibody 12F4 (16 colors) and MAP2 (magenta). Scale bar is equal to 15 μm. E. Quantification of intracellular Aβ 42 levels, as measured by 12F4 intensity, of neurites of ApoE KO, ApoE3-KI and ApoE4-KI primary neurons. Data is shown as mean ± SD. The dashed line indicates the level of unspecific signal detected in APP KO control neurons. * p-value < 0.05. See also Supplementary Figure 5D-E.

    Journal: bioRxiv

    Article Title: Apolipoprotein E intersects with amyloid-β within neurons

    doi: 10.1101/2022.12.13.520238

    Figure Lengend Snippet: A. Schematic visualization of the different primary neuron models used to study the effects of human ApoE isoforms on levels of intraneuronal Aβ 42 . B. Representative epifluorescence images of neuronal cell bodies from ApoE KO, ApoE3-KI and ApoE4-KI primary neurons (19 DIV). The neurons were labeled for endogenous mouse Aβ 42 using antibody 12F4 (16 colors) and MAP2 (magenta). Scale bar is 15 μm. C. Quantification of endogenous Aβ 42 in neuronal cell bodies as shown in . The levels of Aβ 42 were significantly higher in ApoE4-KI neurons compared to ApoE KO neurons. D. Representative images of ApoE KO, ApoE3-KI and ApoE4-KI neurites labeled for Aβ 42 by antibody 12F4 (16 colors) and MAP2 (magenta). Scale bar is equal to 15 μm. E. Quantification of intracellular Aβ 42 levels, as measured by 12F4 intensity, of neurites of ApoE KO, ApoE3-KI and ApoE4-KI primary neurons. Data is shown as mean ± SD. The dashed line indicates the level of unspecific signal detected in APP KO control neurons. * p-value < 0.05. See also Supplementary Figure 5D-E.

    Article Snippet: Recombinant ApoE3 and ApoE4 proteins (Sigma-Aldrich, SRP4696 and A3234, respectively) were reconstituted in 0.1% bovine serum albumin (BSA) in milli-Q water to a stock concentration of 0.1 mg/ml.

    Techniques: Labeling

    A. Schematic representation of 0.5 μM synthetic Aβ 42 treatment to different ApoE neuron cultures: ApoE KO, and human ApoE3- and ApoE4-KI neurons. B. Representative epifluorescence images of neurites from Aβ 42 -treated ApoE KO, ApoE3 and ApoE4 primary neurons. The neurites are labeled for human Aβ antibody 6E10 (red), fibrillar oligomer antibody OC (green) and MAP2 (grey in upper panel). Clear co-localization was observed between 6E10 and OC, suggesting that added synthetic Aβ 42 is aggregating inside neurons. C-D. Quantification of the number of ( C ) and the average area size of the 6E10-positive Aβ 42 puncta ( D ) in Aβ 42 -treated primary neurons as shown in . The number of 6E10 puncta is significantly increased in ApoE4 compared to ApoE3 neurons after Aβ 42 treatment. The quantifications were performed while being blinded ( C-D ). Data is shown as mean ± SD. * < 0.05. px = pixels. See also Supplementary Figure 5F.

    Journal: bioRxiv

    Article Title: Apolipoprotein E intersects with amyloid-β within neurons

    doi: 10.1101/2022.12.13.520238

    Figure Lengend Snippet: A. Schematic representation of 0.5 μM synthetic Aβ 42 treatment to different ApoE neuron cultures: ApoE KO, and human ApoE3- and ApoE4-KI neurons. B. Representative epifluorescence images of neurites from Aβ 42 -treated ApoE KO, ApoE3 and ApoE4 primary neurons. The neurites are labeled for human Aβ antibody 6E10 (red), fibrillar oligomer antibody OC (green) and MAP2 (grey in upper panel). Clear co-localization was observed between 6E10 and OC, suggesting that added synthetic Aβ 42 is aggregating inside neurons. C-D. Quantification of the number of ( C ) and the average area size of the 6E10-positive Aβ 42 puncta ( D ) in Aβ 42 -treated primary neurons as shown in . The number of 6E10 puncta is significantly increased in ApoE4 compared to ApoE3 neurons after Aβ 42 treatment. The quantifications were performed while being blinded ( C-D ). Data is shown as mean ± SD. * < 0.05. px = pixels. See also Supplementary Figure 5F.

    Article Snippet: Recombinant ApoE3 and ApoE4 proteins (Sigma-Aldrich, SRP4696 and A3234, respectively) were reconstituted in 0.1% bovine serum albumin (BSA) in milli-Q water to a stock concentration of 0.1 mg/ml.

    Techniques: Labeling