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  • 94
    Genesee Scientific microtubes, clear
    Microtubes, Clear, supplied by Genesee Scientific, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/microtubes, clear/product/Genesee Scientific
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    microtubes, clear - by Bioz Stars, 2024-02
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    93
    Proteintech 1 ap rrid ab 11183764
    1 Ap Rrid Ab 11183764, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/1 ap rrid ab 11183764/product/Proteintech
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    1 ap rrid ab 11183764 - by Bioz Stars, 2024-02
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    93
    Proteintech anti chc22
    (A) Representative images of GLUT4 (exofacial haemagglutinin (HA)-tag, internal GFP tag) in HeLa-GLUT4 cells before (basal) or after insulin treatment. GLUT4 at the plasma membrane was detected by immunofluorescence (IF) after surface labeling with anti-HA monoclonal antibody (red). Total GLUT4 (green) was detected by GFP tag. Arrows show the GLUT4 storage compartment. Arrowheads point to peripheral GLUT4 vesicles. Scale bars: 7.5 µm. (B) Left panel – Representative FACS histogram of surface GLUT4 fluorescence intensities (signal from anti-HA labeling) before (basal) and after insulin treatment (Ins). Middle panel – Quantification of surface:total GLUT4 (HA:GFP mean fluorescence intensity signals). Data expressed as mean ± SEM, N=3, 10,000 cells acquired per experiment. Two-tailed unpaired Student’s t-test with equal variances, **p<0.01. Right panel – Representative immunoblot for phosphorylated AKT (p-AKT), phosphorylated AS160 (p-AS160), total AS160 and β-actin in HeLa-GLUT4 cells before and after insulin treatment. The migration position of molecular weight (MW) markers is indicated at the left in kilodaltons (kDa). (C) Representative images of total GLUT4 (GFP tag, green) and Major Histocompatibility Complex I molecules (MHCI, blue) before (basal) or after insulin treatment in HeLa-GLUT4 cells transfected with non-targeting control siRNA (siControl) or siRNA targeting <t>CHC22</t> (siCHC22). Scale bars: 8 µm. (D) Representative FACS histograms of surface GLUT4 fluorescence intensity (signal from anti-HA labeling) in HeLa-GLUT4 cells transfected with siControl or siRNA siCHC22 before (red) or after treatment with insulin (blue). Histograms are extracted from the experiment quantified in . (E) Representative IF staining for internalized surface-labeled GLUT4 (HA-tag, blue) and syntaxin 6 (STX-6, red) for HeLa-GLUT4 cells at 0, 10 or 30 minutes after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 7.5 µm. (F) Pearson’s overlap quantification for labeling of STX-6 and HA-tag (Data expressed as mean ± SEM, N=3, 14-19 cells per experiment). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test ****p<0.0001. (G) Left panel – representative Structured Illumination Microscopy (SIM) image of a HeLa-GLUT4 cell stained for STX-6 (red). Total GLUT4 (green) was detected by GFP tag. The gray circle delineates the nucleus (N) and the white square delineates the magnified area displayed in the right panel image. Scale bar: 10 µm. Right panel – the white dashed line in the magnified area spans the segment for which fluorescence intensities for GLUT4 and STX-6 are plotted below, in green and red, respectively. Arrowheads indicate areas of overlap. (H) Representative IF staining for CHC17 (red) and CHC22 (blue) in HeLa-GLUT4 cells transfected with non-targeting siControl or siRNA targeting CHC17 (siCHC17) or siCHC22, with GLUT4 detected by GFP tag (green). Arrows point to a CHC22-depleted cell. Scale bars: 10 µm for siControl and siCHC17 and 7.5 µm for siCHC22. Merged images in (A), (C), (E), (G), (H) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.
    Anti Chc22, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti chc22/product/Proteintech
    Average 93 stars, based on 1 article reviews
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    anti chc22 - by Bioz Stars, 2024-02
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    93
    Proteintech rabbit polyclonal α chc22
    (A) Representative images of GLUT4 (exofacial haemagglutinin (HA)-tag, internal GFP tag) in HeLa-GLUT4 cells before (basal) or after insulin treatment. GLUT4 at the plasma membrane was detected by immunofluorescence (IF) after surface labeling with anti-HA monoclonal antibody (red). Total GLUT4 (green) was detected by GFP tag. Arrows show the GLUT4 storage compartment. Arrowheads point to peripheral GLUT4 vesicles. Scale bars: 7.5 µm. (B) Left panel – Representative FACS histogram of surface GLUT4 fluorescence intensities (signal from anti-HA labeling) before (basal) and after insulin treatment (Ins). Middle panel – Quantification of surface:total GLUT4 (HA:GFP mean fluorescence intensity signals). Data expressed as mean ± SEM, N=3, 10,000 cells acquired per experiment. Two-tailed unpaired Student’s t-test with equal variances, **p<0.01. Right panel – Representative immunoblot for phosphorylated AKT (p-AKT), phosphorylated AS160 (p-AS160), total AS160 and β-actin in HeLa-GLUT4 cells before and after insulin treatment. The migration position of molecular weight (MW) markers is indicated at the left in kilodaltons (kDa). (C) Representative images of total GLUT4 (GFP tag, green) and Major Histocompatibility Complex I molecules (MHCI, blue) before (basal) or after insulin treatment in HeLa-GLUT4 cells transfected with non-targeting control siRNA (siControl) or siRNA targeting <t>CHC22</t> (siCHC22). Scale bars: 8 µm. (D) Representative FACS histograms of surface GLUT4 fluorescence intensity (signal from anti-HA labeling) in HeLa-GLUT4 cells transfected with siControl or siRNA siCHC22 before (red) or after treatment with insulin (blue). Histograms are extracted from the experiment quantified in . (E) Representative IF staining for internalized surface-labeled GLUT4 (HA-tag, blue) and syntaxin 6 (STX-6, red) for HeLa-GLUT4 cells at 0, 10 or 30 minutes after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 7.5 µm. (F) Pearson’s overlap quantification for labeling of STX-6 and HA-tag (Data expressed as mean ± SEM, N=3, 14-19 cells per experiment). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test ****p<0.0001. (G) Left panel – representative Structured Illumination Microscopy (SIM) image of a HeLa-GLUT4 cell stained for STX-6 (red). Total GLUT4 (green) was detected by GFP tag. The gray circle delineates the nucleus (N) and the white square delineates the magnified area displayed in the right panel image. Scale bar: 10 µm. Right panel – the white dashed line in the magnified area spans the segment for which fluorescence intensities for GLUT4 and STX-6 are plotted below, in green and red, respectively. Arrowheads indicate areas of overlap. (H) Representative IF staining for CHC17 (red) and CHC22 (blue) in HeLa-GLUT4 cells transfected with non-targeting siControl or siRNA targeting CHC17 (siCHC17) or siCHC22, with GLUT4 detected by GFP tag (green). Arrows point to a CHC22-depleted cell. Scale bars: 10 µm for siControl and siCHC17 and 7.5 µm for siCHC22. Merged images in (A), (C), (E), (G), (H) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.
    Rabbit Polyclonal α Chc22, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal α chc22/product/Proteintech
    Average 93 stars, based on 1 article reviews
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    rabbit polyclonal α chc22 - by Bioz Stars, 2024-02
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    93
    Proteintech cltc polyclonal antibody
    (A) Representative images of GLUT4 (exofacial haemagglutinin (HA)-tag, internal GFP tag) in HeLa-GLUT4 cells before (basal) or after insulin treatment. GLUT4 at the plasma membrane was detected by immunofluorescence (IF) after surface labeling with anti-HA monoclonal antibody (red). Total GLUT4 (green) was detected by GFP tag. Arrows show the GLUT4 storage compartment. Arrowheads point to peripheral GLUT4 vesicles. Scale bars: 7.5 µm. (B) Left panel – Representative FACS histogram of surface GLUT4 fluorescence intensities (signal from anti-HA labeling) before (basal) and after insulin treatment (Ins). Middle panel – Quantification of surface:total GLUT4 (HA:GFP mean fluorescence intensity signals). Data expressed as mean ± SEM, N=3, 10,000 cells acquired per experiment. Two-tailed unpaired Student’s t-test with equal variances, **p<0.01. Right panel – Representative immunoblot for phosphorylated AKT (p-AKT), phosphorylated AS160 (p-AS160), total AS160 and β-actin in HeLa-GLUT4 cells before and after insulin treatment. The migration position of molecular weight (MW) markers is indicated at the left in kilodaltons (kDa). (C) Representative images of total GLUT4 (GFP tag, green) and Major Histocompatibility Complex I molecules (MHCI, blue) before (basal) or after insulin treatment in HeLa-GLUT4 cells transfected with non-targeting control siRNA (siControl) or siRNA targeting <t>CHC22</t> (siCHC22). Scale bars: 8 µm. (D) Representative FACS histograms of surface GLUT4 fluorescence intensity (signal from anti-HA labeling) in HeLa-GLUT4 cells transfected with siControl or siRNA siCHC22 before (red) or after treatment with insulin (blue). Histograms are extracted from the experiment quantified in . (E) Representative IF staining for internalized surface-labeled GLUT4 (HA-tag, blue) and syntaxin 6 (STX-6, red) for HeLa-GLUT4 cells at 0, 10 or 30 minutes after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 7.5 µm. (F) Pearson’s overlap quantification for labeling of STX-6 and HA-tag (Data expressed as mean ± SEM, N=3, 14-19 cells per experiment). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test ****p<0.0001. (G) Left panel – representative Structured Illumination Microscopy (SIM) image of a HeLa-GLUT4 cell stained for STX-6 (red). Total GLUT4 (green) was detected by GFP tag. The gray circle delineates the nucleus (N) and the white square delineates the magnified area displayed in the right panel image. Scale bar: 10 µm. Right panel – the white dashed line in the magnified area spans the segment for which fluorescence intensities for GLUT4 and STX-6 are plotted below, in green and red, respectively. Arrowheads indicate areas of overlap. (H) Representative IF staining for CHC17 (red) and CHC22 (blue) in HeLa-GLUT4 cells transfected with non-targeting siControl or siRNA targeting CHC17 (siCHC17) or siCHC22, with GLUT4 detected by GFP tag (green). Arrows point to a CHC22-depleted cell. Scale bars: 10 µm for siControl and siCHC17 and 7.5 µm for siCHC22. Merged images in (A), (C), (E), (G), (H) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.
    Cltc Polyclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cltc polyclonal antibody/product/Proteintech
    Average 93 stars, based on 1 article reviews
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    cltc polyclonal antibody - by Bioz Stars, 2024-02
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    Image Search Results


    (A) Representative images of GLUT4 (exofacial haemagglutinin (HA)-tag, internal GFP tag) in HeLa-GLUT4 cells before (basal) or after insulin treatment. GLUT4 at the plasma membrane was detected by immunofluorescence (IF) after surface labeling with anti-HA monoclonal antibody (red). Total GLUT4 (green) was detected by GFP tag. Arrows show the GLUT4 storage compartment. Arrowheads point to peripheral GLUT4 vesicles. Scale bars: 7.5 µm. (B) Left panel – Representative FACS histogram of surface GLUT4 fluorescence intensities (signal from anti-HA labeling) before (basal) and after insulin treatment (Ins). Middle panel – Quantification of surface:total GLUT4 (HA:GFP mean fluorescence intensity signals). Data expressed as mean ± SEM, N=3, 10,000 cells acquired per experiment. Two-tailed unpaired Student’s t-test with equal variances, **p<0.01. Right panel – Representative immunoblot for phosphorylated AKT (p-AKT), phosphorylated AS160 (p-AS160), total AS160 and β-actin in HeLa-GLUT4 cells before and after insulin treatment. The migration position of molecular weight (MW) markers is indicated at the left in kilodaltons (kDa). (C) Representative images of total GLUT4 (GFP tag, green) and Major Histocompatibility Complex I molecules (MHCI, blue) before (basal) or after insulin treatment in HeLa-GLUT4 cells transfected with non-targeting control siRNA (siControl) or siRNA targeting CHC22 (siCHC22). Scale bars: 8 µm. (D) Representative FACS histograms of surface GLUT4 fluorescence intensity (signal from anti-HA labeling) in HeLa-GLUT4 cells transfected with siControl or siRNA siCHC22 before (red) or after treatment with insulin (blue). Histograms are extracted from the experiment quantified in . (E) Representative IF staining for internalized surface-labeled GLUT4 (HA-tag, blue) and syntaxin 6 (STX-6, red) for HeLa-GLUT4 cells at 0, 10 or 30 minutes after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 7.5 µm. (F) Pearson’s overlap quantification for labeling of STX-6 and HA-tag (Data expressed as mean ± SEM, N=3, 14-19 cells per experiment). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test ****p<0.0001. (G) Left panel – representative Structured Illumination Microscopy (SIM) image of a HeLa-GLUT4 cell stained for STX-6 (red). Total GLUT4 (green) was detected by GFP tag. The gray circle delineates the nucleus (N) and the white square delineates the magnified area displayed in the right panel image. Scale bar: 10 µm. Right panel – the white dashed line in the magnified area spans the segment for which fluorescence intensities for GLUT4 and STX-6 are plotted below, in green and red, respectively. Arrowheads indicate areas of overlap. (H) Representative IF staining for CHC17 (red) and CHC22 (blue) in HeLa-GLUT4 cells transfected with non-targeting siControl or siRNA targeting CHC17 (siCHC17) or siCHC22, with GLUT4 detected by GFP tag (green). Arrows point to a CHC22-depleted cell. Scale bars: 10 µm for siControl and siCHC17 and 7.5 µm for siCHC22. Merged images in (A), (C), (E), (G), (H) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: (A) Representative images of GLUT4 (exofacial haemagglutinin (HA)-tag, internal GFP tag) in HeLa-GLUT4 cells before (basal) or after insulin treatment. GLUT4 at the plasma membrane was detected by immunofluorescence (IF) after surface labeling with anti-HA monoclonal antibody (red). Total GLUT4 (green) was detected by GFP tag. Arrows show the GLUT4 storage compartment. Arrowheads point to peripheral GLUT4 vesicles. Scale bars: 7.5 µm. (B) Left panel – Representative FACS histogram of surface GLUT4 fluorescence intensities (signal from anti-HA labeling) before (basal) and after insulin treatment (Ins). Middle panel – Quantification of surface:total GLUT4 (HA:GFP mean fluorescence intensity signals). Data expressed as mean ± SEM, N=3, 10,000 cells acquired per experiment. Two-tailed unpaired Student’s t-test with equal variances, **p<0.01. Right panel – Representative immunoblot for phosphorylated AKT (p-AKT), phosphorylated AS160 (p-AS160), total AS160 and β-actin in HeLa-GLUT4 cells before and after insulin treatment. The migration position of molecular weight (MW) markers is indicated at the left in kilodaltons (kDa). (C) Representative images of total GLUT4 (GFP tag, green) and Major Histocompatibility Complex I molecules (MHCI, blue) before (basal) or after insulin treatment in HeLa-GLUT4 cells transfected with non-targeting control siRNA (siControl) or siRNA targeting CHC22 (siCHC22). Scale bars: 8 µm. (D) Representative FACS histograms of surface GLUT4 fluorescence intensity (signal from anti-HA labeling) in HeLa-GLUT4 cells transfected with siControl or siRNA siCHC22 before (red) or after treatment with insulin (blue). Histograms are extracted from the experiment quantified in . (E) Representative IF staining for internalized surface-labeled GLUT4 (HA-tag, blue) and syntaxin 6 (STX-6, red) for HeLa-GLUT4 cells at 0, 10 or 30 minutes after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 7.5 µm. (F) Pearson’s overlap quantification for labeling of STX-6 and HA-tag (Data expressed as mean ± SEM, N=3, 14-19 cells per experiment). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test ****p<0.0001. (G) Left panel – representative Structured Illumination Microscopy (SIM) image of a HeLa-GLUT4 cell stained for STX-6 (red). Total GLUT4 (green) was detected by GFP tag. The gray circle delineates the nucleus (N) and the white square delineates the magnified area displayed in the right panel image. Scale bar: 10 µm. Right panel – the white dashed line in the magnified area spans the segment for which fluorescence intensities for GLUT4 and STX-6 are plotted below, in green and red, respectively. Arrowheads indicate areas of overlap. (H) Representative IF staining for CHC17 (red) and CHC22 (blue) in HeLa-GLUT4 cells transfected with non-targeting siControl or siRNA targeting CHC17 (siCHC17) or siCHC22, with GLUT4 detected by GFP tag (green). Arrows point to a CHC22-depleted cell. Scale bars: 10 µm for siControl and siCHC17 and 7.5 µm for siCHC22. Merged images in (A), (C), (E), (G), (H) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Immunofluorescence, Labeling, Fluorescence, Two Tailed Test, Western Blot, Migration, Molecular Weight, Transfection, Staining, Microscopy

    (A) Representative stills extracted from Video 1 showing a HeLa cell expressing the endoplasmic reticulum (ER) Ii-hook fused to streptavidin along with HA-GLUT1-SBP-mCherry (GLUT1, red) and HA-GLUT4-SBP-GFP (GLUT4, green). The intracellular traffic of GLUT1-mCherry and GLUT4-GFP was simultaneously tracked for 1h after biotin addition released them from the ER. Upon ER exit, both GLUT1 and GLUT4 accumulated in the perinuclear region of the cell (yellow). From 26 min onwards, highly mobile GLUT1 vesicles (arrowheads) were visible (red) while GLUT4 remained perinuclear. Scale bar: 10 µm. (B, D, F, H, J) Representative immunofluorescence staining for GLUT1-SBP-GFP or GLUT4-SBP-GFP (detected with anti-GFP antibody, green), CHC22 (red) and (B) calnexin (CNX, blue), (D) ERGIC-53 (blue), (F) p115 (blue), (H) GM130 (blue) or (J) TGN46 (blue) in HeLa cells expressing HA-GLUT1-SBP-GFP or HA-GLUT4-SBP-GFP along with the ER Ii-hook. Traffic of GLUT4 and GLUT1 was tracked at 0, 15, 30 and 60 minutes after release from the ER by biotin. Arrows point to GLUT1 detected at the plasma membrane and arrowheads point to GLUT1-positive endosomal structures. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white. Scale bars: 10 µm. (C, E, G, I, K, L) Pearson’s overlap between GLUT1 or GLUT4 and CNX, ERGIC-53, p115, GM130, TGN46 or CHC22 at different time-points post-ER release. Data expressed as mean ± SEM, N=3-4, 10-46 cells per experiment. One-way analysis of variance (ANOVA) followed by Sidak’s multiple comparison post-hoc test *p<0.05, **p<0.01, ****p<0.0001 to test differences between GLUT1 and GLUT4 overlap with markers at each time points. (M) Pearson’s overlap between CHC22 and GLUT4, ER marker calreticulin, ERGIC markers p115 and ERGIC-53, cis-Golgi marker GM130 or trans-Golgi marker TGN46 in HeLa-GLUT4 from images taken by confocal microscopy (corresponding representative immunofluorescence staining in ). Data expressed as mean ± SEM, N=3, 4-10 cells across 3 independent samples.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: (A) Representative stills extracted from Video 1 showing a HeLa cell expressing the endoplasmic reticulum (ER) Ii-hook fused to streptavidin along with HA-GLUT1-SBP-mCherry (GLUT1, red) and HA-GLUT4-SBP-GFP (GLUT4, green). The intracellular traffic of GLUT1-mCherry and GLUT4-GFP was simultaneously tracked for 1h after biotin addition released them from the ER. Upon ER exit, both GLUT1 and GLUT4 accumulated in the perinuclear region of the cell (yellow). From 26 min onwards, highly mobile GLUT1 vesicles (arrowheads) were visible (red) while GLUT4 remained perinuclear. Scale bar: 10 µm. (B, D, F, H, J) Representative immunofluorescence staining for GLUT1-SBP-GFP or GLUT4-SBP-GFP (detected with anti-GFP antibody, green), CHC22 (red) and (B) calnexin (CNX, blue), (D) ERGIC-53 (blue), (F) p115 (blue), (H) GM130 (blue) or (J) TGN46 (blue) in HeLa cells expressing HA-GLUT1-SBP-GFP or HA-GLUT4-SBP-GFP along with the ER Ii-hook. Traffic of GLUT4 and GLUT1 was tracked at 0, 15, 30 and 60 minutes after release from the ER by biotin. Arrows point to GLUT1 detected at the plasma membrane and arrowheads point to GLUT1-positive endosomal structures. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white. Scale bars: 10 µm. (C, E, G, I, K, L) Pearson’s overlap between GLUT1 or GLUT4 and CNX, ERGIC-53, p115, GM130, TGN46 or CHC22 at different time-points post-ER release. Data expressed as mean ± SEM, N=3-4, 10-46 cells per experiment. One-way analysis of variance (ANOVA) followed by Sidak’s multiple comparison post-hoc test *p<0.05, **p<0.01, ****p<0.0001 to test differences between GLUT1 and GLUT4 overlap with markers at each time points. (M) Pearson’s overlap between CHC22 and GLUT4, ER marker calreticulin, ERGIC markers p115 and ERGIC-53, cis-Golgi marker GM130 or trans-Golgi marker TGN46 in HeLa-GLUT4 from images taken by confocal microscopy (corresponding representative immunofluorescence staining in ). Data expressed as mean ± SEM, N=3, 4-10 cells across 3 independent samples.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Expressing, Immunofluorescence, Staining, Marker, Confocal Microscopy

    (A) CHC17 (X22 antibody) or CHC22 (CLTCL1 antibody from Proteintech) immunoblots (IB) of clathrin coated vesicles (CCV) purified from pig brain containing only CHC17 or of cell lysate from bacteria expressing low levels of the hub fragment (residues 1074-1640) of CHC22 (hub 22). The migration of molecular weight (MW) markers is indicated in kilodaltons (kDa). Ponceau staining for proteins is shown on the right (Pro). (B) Representative confocal microscopy immunofluorescence (IF) imaging of CHC22 (red or blue), p115 (red or blue) and GLUT4 (green) in HeLa-GLUT4 cells (top panel) or LHCNM2 myotubes (bottom panel). (C) Representative IF staining for CHC22 (blue), ERGIC-53 (red) and GLUT4 (green) in HeLa-GLUT4 cells (top panel) or LHCNM2 myotubes (bottom panel). Scale bars: 5 µm for HeLa-GLUT4 cells and 7.5 µm for LHCNM2 myotubes in (B) and (C). (D) Representative IF staining for CHC22 (blue), GM130 or TGN46 (red) and GLUT4 (GFP, green) in HeLa-GLUT4 cells. Scale bars: 5 µm. (E) Representative IF staining for CHC22 (blue), GM130 or TGN46 (green) and p115 (red) in LHCNM2 myotubes. Scale bars: 7.5 µm. (F) Representative IF staining for CHC22 (blue), calreticulin (red) and GLUT4 (green) in HeLa-GLUT4 cells. Scale bars: 5 µm. (G) Representative IF staining for CHC22 (red), calnexin (CNX, blue) and GLUT4 (green) in hSkMC-AB1190-GLUT4. Scale bars: 10 µm. (H) Representative Structured Illumination Microscopy (SIM) of a HeLa-GLUT4 (HeLa-G4) cell (top panel) and human skeletal muscle cell (hSkMC-AB1190-GLUT4, bottom panel) stained for CHC22 (red) and TGN46 (blue). GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 and immunostained with an anti-GFP antibody in hSkMC-AB1190-GLUT4. Scale bar: 10 µm. Merged images in (B-H) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white. (I) Representative fluorescence intensity plots for GLUT4 (green), CHC22 (red) and p115, ERGIC-53, GM130 or TGN46 (blue) generated from SIM images of the perinuclear region of HeLa-GLUT4 cells.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: (A) CHC17 (X22 antibody) or CHC22 (CLTCL1 antibody from Proteintech) immunoblots (IB) of clathrin coated vesicles (CCV) purified from pig brain containing only CHC17 or of cell lysate from bacteria expressing low levels of the hub fragment (residues 1074-1640) of CHC22 (hub 22). The migration of molecular weight (MW) markers is indicated in kilodaltons (kDa). Ponceau staining for proteins is shown on the right (Pro). (B) Representative confocal microscopy immunofluorescence (IF) imaging of CHC22 (red or blue), p115 (red or blue) and GLUT4 (green) in HeLa-GLUT4 cells (top panel) or LHCNM2 myotubes (bottom panel). (C) Representative IF staining for CHC22 (blue), ERGIC-53 (red) and GLUT4 (green) in HeLa-GLUT4 cells (top panel) or LHCNM2 myotubes (bottom panel). Scale bars: 5 µm for HeLa-GLUT4 cells and 7.5 µm for LHCNM2 myotubes in (B) and (C). (D) Representative IF staining for CHC22 (blue), GM130 or TGN46 (red) and GLUT4 (GFP, green) in HeLa-GLUT4 cells. Scale bars: 5 µm. (E) Representative IF staining for CHC22 (blue), GM130 or TGN46 (green) and p115 (red) in LHCNM2 myotubes. Scale bars: 7.5 µm. (F) Representative IF staining for CHC22 (blue), calreticulin (red) and GLUT4 (green) in HeLa-GLUT4 cells. Scale bars: 5 µm. (G) Representative IF staining for CHC22 (red), calnexin (CNX, blue) and GLUT4 (green) in hSkMC-AB1190-GLUT4. Scale bars: 10 µm. (H) Representative Structured Illumination Microscopy (SIM) of a HeLa-GLUT4 (HeLa-G4) cell (top panel) and human skeletal muscle cell (hSkMC-AB1190-GLUT4, bottom panel) stained for CHC22 (red) and TGN46 (blue). GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 and immunostained with an anti-GFP antibody in hSkMC-AB1190-GLUT4. Scale bar: 10 µm. Merged images in (B-H) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white. (I) Representative fluorescence intensity plots for GLUT4 (green), CHC22 (red) and p115, ERGIC-53, GM130 or TGN46 (blue) generated from SIM images of the perinuclear region of HeLa-GLUT4 cells.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Western Blot, Purification, Expressing, Migration, Molecular Weight, Staining, Confocal Microscopy, Immunofluorescence, Imaging, Microscopy, Fluorescence, Generated

    (A, B) Representative immunofluorescence (IF) staining for CHC22 (blue) and p115 (red) in HeLa-GLUT4 cells (A) or LHCNM2 myotubes (B) treated or not with Brefeldin A (BFA). GLUT4 (green) was detected by GFP tag in HeLa-G4 cells and by IF of endogenous protein in LHCNM2 cells. Scale bars: 5 and 25 µm for (A) and (B), respectively. (C, D) Representative immunofluorescence (IF) staining of HeLa-GLUT4 cells for CHC22 (blue) and ERGIC-53 (red) in (C) or Rab1 (red) in (D) treated or not with BFA and stimulated or not by insulin (Ins). GLUT4 (green) was detected by GFP. Scale bars: 10 µm. (E) Quantification of Pearson’s overlap values between CHC22, GLUT4, ERGIC-53 and Rab1 (data as in (C & D) expressed as mean ± SEM, N=3 to 4, 5 to 42 cells per experiment). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test *p<0.05, ***p<0.001. (F) Representative immunofluorescence staining for CHC22 (blue), ERGIC-53 (red) and GLUT4 (anti-GFP antibody, green) in hSkMC-AB1190-GLUT4 treated or not with Brefeldin A (BFA) and stimulated or not by insulin (Ins). Scale bar: 10 µm. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: (A, B) Representative immunofluorescence (IF) staining for CHC22 (blue) and p115 (red) in HeLa-GLUT4 cells (A) or LHCNM2 myotubes (B) treated or not with Brefeldin A (BFA). GLUT4 (green) was detected by GFP tag in HeLa-G4 cells and by IF of endogenous protein in LHCNM2 cells. Scale bars: 5 and 25 µm for (A) and (B), respectively. (C, D) Representative immunofluorescence (IF) staining of HeLa-GLUT4 cells for CHC22 (blue) and ERGIC-53 (red) in (C) or Rab1 (red) in (D) treated or not with BFA and stimulated or not by insulin (Ins). GLUT4 (green) was detected by GFP. Scale bars: 10 µm. (E) Quantification of Pearson’s overlap values between CHC22, GLUT4, ERGIC-53 and Rab1 (data as in (C & D) expressed as mean ± SEM, N=3 to 4, 5 to 42 cells per experiment). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test *p<0.05, ***p<0.001. (F) Representative immunofluorescence staining for CHC22 (blue), ERGIC-53 (red) and GLUT4 (anti-GFP antibody, green) in hSkMC-AB1190-GLUT4 treated or not with Brefeldin A (BFA) and stimulated or not by insulin (Ins). Scale bar: 10 µm. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Immunofluorescence, Staining

    Representative immunofluorescence images of single A549 cells from cultures treated with siRNA targeting CHC22 or with non-targeting control siRNA and labeled for Sec22b (red) and CHC22 (green), 1h post-infection with wild-type L.p. (MOI=50). Arrows point to L.p. detected with DAPI. Boxed inserts show L.p. region at 5X and 2X magnification for siControl and siCHC22, respectively. Scale bar: 5 µm referring to main images. (B) Quantification of the proportion of L.p. vacuoles staining positive for Sec22b. Data expressed as mean ± SEM, N=4, 10 to 20 vacuoles counted per experiment as represented in (A). Two-tailed unpaired Student’s t-test with equal variances ***p<0.001. (C) Representative images of HeLa cells transiently expressing FcγRII (needed for L.p. infection) , infected with wild type (WT) or mutant ΔdotA L.p. (MOI=50, red) and labeled 1 hour post-infection with antibodies against Rab1 (green). Hoechst stains the nuclei (blue). Arrows point to L.p. , Scale bar: 10 µm. (D) Representative images of A549 cells transiently transfected with HA-GLUT4-GFP (green), infected with wild type L.p. expressing mono-DsRed protein ( L.p.- DsRed, MOI=50, red) and labeled 1 hour post-infection with antibodies against endogenous CHC17 (upper panel) or CHC22 (lower panel) (blue). Scale bars: 5 µm. (E) Representative images of HeLa cells transiently expressing FcγRII, infected with wild type (WT) or mutant ΔdotA L.p. (MOI=50, red) and labeled 1 hour post-infection with antibodies against Sec16a (E) (green). Hoechst stains the nuclei (blue). Arrows point to L.p. , Scale bar: 10 µm. (F) Quantification of the proportion of vacuoles staining positive for Sec16a. Data expressed as mean ± SEM, N=3, 50 vacuoles counted per experiment as represented in (E). Merged images in (A and D) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white. Merged images in (C and E) show red/green overlap in yellow.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: Representative immunofluorescence images of single A549 cells from cultures treated with siRNA targeting CHC22 or with non-targeting control siRNA and labeled for Sec22b (red) and CHC22 (green), 1h post-infection with wild-type L.p. (MOI=50). Arrows point to L.p. detected with DAPI. Boxed inserts show L.p. region at 5X and 2X magnification for siControl and siCHC22, respectively. Scale bar: 5 µm referring to main images. (B) Quantification of the proportion of L.p. vacuoles staining positive for Sec22b. Data expressed as mean ± SEM, N=4, 10 to 20 vacuoles counted per experiment as represented in (A). Two-tailed unpaired Student’s t-test with equal variances ***p<0.001. (C) Representative images of HeLa cells transiently expressing FcγRII (needed for L.p. infection) , infected with wild type (WT) or mutant ΔdotA L.p. (MOI=50, red) and labeled 1 hour post-infection with antibodies against Rab1 (green). Hoechst stains the nuclei (blue). Arrows point to L.p. , Scale bar: 10 µm. (D) Representative images of A549 cells transiently transfected with HA-GLUT4-GFP (green), infected with wild type L.p. expressing mono-DsRed protein ( L.p.- DsRed, MOI=50, red) and labeled 1 hour post-infection with antibodies against endogenous CHC17 (upper panel) or CHC22 (lower panel) (blue). Scale bars: 5 µm. (E) Representative images of HeLa cells transiently expressing FcγRII, infected with wild type (WT) or mutant ΔdotA L.p. (MOI=50, red) and labeled 1 hour post-infection with antibodies against Sec16a (E) (green). Hoechst stains the nuclei (blue). Arrows point to L.p. , Scale bar: 10 µm. (F) Quantification of the proportion of vacuoles staining positive for Sec16a. Data expressed as mean ± SEM, N=3, 50 vacuoles counted per experiment as represented in (E). Merged images in (A and D) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white. Merged images in (C and E) show red/green overlap in yellow.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Immunofluorescence, Labeling, Infection, Staining, Two Tailed Test, Expressing, Mutagenesis, Transfection

    (A and B) Representative Structured Illumination Microscopy of a HeLa-GLUT4 cell (HeLa-G4) (A) and the human skeletal muscle cell line hSkMC-AB1190-GLUT4 (hSkMC) (B) stained for CHC22 (red) and p115 (blue). The gray circles delineate the nuclei (N). Muscle cell staining with each antibody is shown in black on white below the color images. Scale bars: 10 µm. (C and D) Representative Structured Illumination Microscopy of the perinuclear region of HeLa-GLUT4 cells and hSkMC-AB1190-GLUT4 stained for CHC22 (red) and p115 (C), ERGIC-53 (blue) (D). The solid gray lines delineate the nuclear border (N). The dashed white lines span the segment for which fluorescence intensities for GLUT4 (green), CHC22 (red) and p115, ERGIC-53 (blue) were plotted. Arrowheads indicate areas of peak overlap. Scale bars: 1 µm. In A, B, C and D, GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 or immunostained with anti-GFP antibody in hSkMC-AB1190-GLUT4. Merged images in (A, B and C) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: (A and B) Representative Structured Illumination Microscopy of a HeLa-GLUT4 cell (HeLa-G4) (A) and the human skeletal muscle cell line hSkMC-AB1190-GLUT4 (hSkMC) (B) stained for CHC22 (red) and p115 (blue). The gray circles delineate the nuclei (N). Muscle cell staining with each antibody is shown in black on white below the color images. Scale bars: 10 µm. (C and D) Representative Structured Illumination Microscopy of the perinuclear region of HeLa-GLUT4 cells and hSkMC-AB1190-GLUT4 stained for CHC22 (red) and p115 (C), ERGIC-53 (blue) (D). The solid gray lines delineate the nuclear border (N). The dashed white lines span the segment for which fluorescence intensities for GLUT4 (green), CHC22 (red) and p115, ERGIC-53 (blue) were plotted. Arrowheads indicate areas of peak overlap. Scale bars: 1 µm. In A, B, C and D, GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 or immunostained with anti-GFP antibody in hSkMC-AB1190-GLUT4. Merged images in (A, B and C) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Microscopy, Staining, Fluorescence

    (A, B and C) Representative Structured Illumination Microscopy of the perinuclear region of HeLa-GLUT4 cells (HeLa-G4) and hSkMC-AB1190-GLUT4 (hSkMC) stained for CHC22 (red) and GM130 (A), TGN46 (B) and syntaxin 6 (STX-6, blue) (C). The solid gray lines delineate the nuclear border (N). The dashed white lines span the segment over which fluorescence intensities for GLUT4 (green), CHC22 (red) and GM130, TGN46 and STX-6 (blue) were plotted. Scale bars: 1 µm. In A, B and C, GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 in HeLa-GLUT4 or immunostained using an anti-GFP antibody in hSkMC-AB1190-GLUT4. Merged images in (A, B and C) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: (A, B and C) Representative Structured Illumination Microscopy of the perinuclear region of HeLa-GLUT4 cells (HeLa-G4) and hSkMC-AB1190-GLUT4 (hSkMC) stained for CHC22 (red) and GM130 (A), TGN46 (B) and syntaxin 6 (STX-6, blue) (C). The solid gray lines delineate the nuclear border (N). The dashed white lines span the segment over which fluorescence intensities for GLUT4 (green), CHC22 (red) and GM130, TGN46 and STX-6 (blue) were plotted. Scale bars: 1 µm. In A, B and C, GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 in HeLa-GLUT4 or immunostained using an anti-GFP antibody in hSkMC-AB1190-GLUT4. Merged images in (A, B and C) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Microscopy, Staining, Fluorescence

    (A) Representative immunofluorescence (IF) staining for internalized surface-labeled GLUT4 (HA-tag, blue) and CHC22 (red) for HeLa-GLUT4 cells at 0, 10 or 30 minutes after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 5 µm. (B) Pearson’s overlap for labeling of CHC22 and HA-tag (data expressed as mean ± SEM, N=3, 8-40 cells per experiment). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test *p<0.05, ****p<0.0001. (C) Representative immunofluorescence (IF) staining for internalized surface-labeled GLUT4 (HA-tag, blue) and ERGIC-53 (red) for HeLa-GLUT4 cells at 0, 10 or 30 minutes after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 10 µm. (D) Pearson’s overlap for labeling of ERGIC-53 and HA-tag (data expressed as mean ± SEM, N=3, 14-22 cells per experiment). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test ****p<0.0001. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: (A) Representative immunofluorescence (IF) staining for internalized surface-labeled GLUT4 (HA-tag, blue) and CHC22 (red) for HeLa-GLUT4 cells at 0, 10 or 30 minutes after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 5 µm. (B) Pearson’s overlap for labeling of CHC22 and HA-tag (data expressed as mean ± SEM, N=3, 8-40 cells per experiment). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test *p<0.05, ****p<0.0001. (C) Representative immunofluorescence (IF) staining for internalized surface-labeled GLUT4 (HA-tag, blue) and ERGIC-53 (red) for HeLa-GLUT4 cells at 0, 10 or 30 minutes after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 10 µm. (D) Pearson’s overlap for labeling of ERGIC-53 and HA-tag (data expressed as mean ± SEM, N=3, 14-22 cells per experiment). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test ****p<0.0001. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Immunofluorescence, Staining, Labeling

    (A) Representative images of Legionella pneumophila ( L.p. )-infected A549 cells transiently transfected with GFP-tagged CHC22 or CHC17 (green). One hour after infection with either wild type (WT) or mutant ΔdotA L.p. (MOI=50), bacteria were detected by immunofluorescence (IF, red). Arrows point to L.p. and boxed inserts (upper right or left) show L.p. region at 5X magnification. Scale bars: 10 µm for cells expressing CHC22-GFP and 7.5 µm for cells expressing CHC17-GFP. (B) Quantification of the proportion of L.p. vacuoles positive for CHC22 or CHC17. Data expressed as mean ± SEM, N=3, 4 to 35 vacuoles counted per experiment performed as represented in (A). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test ***p<0.001. (C) Representative images of A549 cells infected with WT L.p. (MOI=50) immunolabeled for endogenous CHC22 or CHC17 (red) and L.p. (green) by IF. Arrows point to L.p. , dashed lines delineate cell borders. Scale bar: 5 µm. (D) Quantification of the proportion of replicative vacuoles (8 hours post-infection) containing 1, 2 to 4, or more than 4 WT or ΔdotA L.p. after treatment with siRNA targeting CHC22 (siCHC22) or CHC17 (siCHC17) or non-targeting siRNA (siControl). Data expressed as mean ± SEM, N=3, over 140 vacuoles counted per experiment. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test was performed to compare the number of cells with a vacuole containing more than 4 bacteria. ****p<0.0001 versus siControl-transfected cells infected with WT L.p. . ++++p<0.0001 versus siControl-transfected cells infected with ΔdotA L.p. (E) Quantification of the proportion of L.p. vacuoles positive for GLUT4-GFP, p115, GGA2, sortilin, IRAP or Rab1 1h after infection with WT or ΔdotA L.p. in HeLa cells transiently expressing FcγRII (needed for L.p. infection) . Data expressed as mean ± SEM, N=3, 4 to 50 vacuoles counted per experiment. Two-tailed unpaired Student’s t-test with equal variances: *p<0.05, **p<0.01, ***p<0.001. (F-I) Representative immunofluorescence of HeLa cells one hour after infection with either wild type (WT) or mutant ΔdotA L.p. (MOI=50) stained for L.p. (red) and sortilin (F), IRAP (G), GGA2 (I), or p115 (green) (I). Hoechst stains the nuclei (blue). Arrows point to L.p. -containing vacuoles, Scale bars: 10 µm. Merged images in (A), (C), (F-I) show red/green overlap in yellow.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: (A) Representative images of Legionella pneumophila ( L.p. )-infected A549 cells transiently transfected with GFP-tagged CHC22 or CHC17 (green). One hour after infection with either wild type (WT) or mutant ΔdotA L.p. (MOI=50), bacteria were detected by immunofluorescence (IF, red). Arrows point to L.p. and boxed inserts (upper right or left) show L.p. region at 5X magnification. Scale bars: 10 µm for cells expressing CHC22-GFP and 7.5 µm for cells expressing CHC17-GFP. (B) Quantification of the proportion of L.p. vacuoles positive for CHC22 or CHC17. Data expressed as mean ± SEM, N=3, 4 to 35 vacuoles counted per experiment performed as represented in (A). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test ***p<0.001. (C) Representative images of A549 cells infected with WT L.p. (MOI=50) immunolabeled for endogenous CHC22 or CHC17 (red) and L.p. (green) by IF. Arrows point to L.p. , dashed lines delineate cell borders. Scale bar: 5 µm. (D) Quantification of the proportion of replicative vacuoles (8 hours post-infection) containing 1, 2 to 4, or more than 4 WT or ΔdotA L.p. after treatment with siRNA targeting CHC22 (siCHC22) or CHC17 (siCHC17) or non-targeting siRNA (siControl). Data expressed as mean ± SEM, N=3, over 140 vacuoles counted per experiment. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test was performed to compare the number of cells with a vacuole containing more than 4 bacteria. ****p<0.0001 versus siControl-transfected cells infected with WT L.p. . ++++p<0.0001 versus siControl-transfected cells infected with ΔdotA L.p. (E) Quantification of the proportion of L.p. vacuoles positive for GLUT4-GFP, p115, GGA2, sortilin, IRAP or Rab1 1h after infection with WT or ΔdotA L.p. in HeLa cells transiently expressing FcγRII (needed for L.p. infection) . Data expressed as mean ± SEM, N=3, 4 to 50 vacuoles counted per experiment. Two-tailed unpaired Student’s t-test with equal variances: *p<0.05, **p<0.01, ***p<0.001. (F-I) Representative immunofluorescence of HeLa cells one hour after infection with either wild type (WT) or mutant ΔdotA L.p. (MOI=50) stained for L.p. (red) and sortilin (F), IRAP (G), GGA2 (I), or p115 (green) (I). Hoechst stains the nuclei (blue). Arrows point to L.p. -containing vacuoles, Scale bars: 10 µm. Merged images in (A), (C), (F-I) show red/green overlap in yellow.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Infection, Transfection, Mutagenesis, Immunofluorescence, Expressing, Immunolabeling, Two Tailed Test, Staining

    (A-D) Representative immunoblots of immunoprecipitates of CHC22, CHC17 (A-D) or Sortilin (C and D) from HeLa-GLUT4 cells (A and C) and hSKMC-AB1190 (B and D) immunoblotted for CHC22, CHC17, p115, GLUT4 and Sortilin. The position of molecular weight (MW) markers is indicated in kilodaltons (kDa). (E-G) Representative immunoblots of HeLa-GLUT4 cells transfected with siRNA targeting CHC22, CHC17 (E, F) or p115 (F, G) or with non-targeting control siRNA (40 nM for 72h) showing levels of CHC22, GLUT4, GGA2, CHC17, p115, sortilin and β-actin (E, G) or levels of IRAP and β-actin (F). The position of molecular weight (MW) markers is indicated in kilodaltons (kDa). (H-J) Quantifications of immunoblot signals as shown in (E-G). Blot signals were normalized to β-actin for each experiment and the fold change (negative values indicate decrease and positive values indicate increase) relative to the normalized signal in control siRNA-treated cell lysates is plotted. Data expressed as mean ± SEM, N=7-8. Two-tailed unpaired Student’s t-test, with Welch’s correction where variances were unequal: *p<0.05; ***p<0.001, ****p<0.0001.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: (A-D) Representative immunoblots of immunoprecipitates of CHC22, CHC17 (A-D) or Sortilin (C and D) from HeLa-GLUT4 cells (A and C) and hSKMC-AB1190 (B and D) immunoblotted for CHC22, CHC17, p115, GLUT4 and Sortilin. The position of molecular weight (MW) markers is indicated in kilodaltons (kDa). (E-G) Representative immunoblots of HeLa-GLUT4 cells transfected with siRNA targeting CHC22, CHC17 (E, F) or p115 (F, G) or with non-targeting control siRNA (40 nM for 72h) showing levels of CHC22, GLUT4, GGA2, CHC17, p115, sortilin and β-actin (E, G) or levels of IRAP and β-actin (F). The position of molecular weight (MW) markers is indicated in kilodaltons (kDa). (H-J) Quantifications of immunoblot signals as shown in (E-G). Blot signals were normalized to β-actin for each experiment and the fold change (negative values indicate decrease and positive values indicate increase) relative to the normalized signal in control siRNA-treated cell lysates is plotted. Data expressed as mean ± SEM, N=7-8. Two-tailed unpaired Student’s t-test, with Welch’s correction where variances were unequal: *p<0.05; ***p<0.001, ****p<0.0001.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Western Blot, Molecular Weight, Transfection, Two Tailed Test

    (A) Immunoblotting for CHC22, CHC17, p115, GM130 and β-actin after transfection of HeLa-GLUT4 cells with siRNA targeting CHC17, CHC22, p115, GM130, sortilin, IRAP or with non-targeting control siRNA. The position of molecular weight (MW) markers is indicated in kilodaltons (kDa). (B) Representative immunofluorescence (IF) staining for CHC22 (red) and p115 (blue) in HeLa-GLUT4 cells after siRNA transfection as in (A), with GLUT4 detected by GFP tag (green). N, nuclei. Arrow points to CHC22-depleted cell. Scale bars: 10 µm. (C) Representative IF staining for GM130 (yellow), p115 (red) and CHC22 (blue) in HeLa-GLUT4 cells after treatment with siRNA targeting GM130 or with control siRNA, with GLUT4 detected by GFP tag (green). Individual antibody staining is shown in black and white, while the merged image shows all four colors with overlaps in white. Scale bars: 25 µm. (D and E) Representative IF staining for GLUT4 (green), CHC22 or sortilin (red) and p115 (blue) in HeLa-GLUT4 cells after treatment with siRNA targeting sortilin or IRAP or with non-targeting control. Scale bars: 5 µm. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: (A) Immunoblotting for CHC22, CHC17, p115, GM130 and β-actin after transfection of HeLa-GLUT4 cells with siRNA targeting CHC17, CHC22, p115, GM130, sortilin, IRAP or with non-targeting control siRNA. The position of molecular weight (MW) markers is indicated in kilodaltons (kDa). (B) Representative immunofluorescence (IF) staining for CHC22 (red) and p115 (blue) in HeLa-GLUT4 cells after siRNA transfection as in (A), with GLUT4 detected by GFP tag (green). N, nuclei. Arrow points to CHC22-depleted cell. Scale bars: 10 µm. (C) Representative IF staining for GM130 (yellow), p115 (red) and CHC22 (blue) in HeLa-GLUT4 cells after treatment with siRNA targeting GM130 or with control siRNA, with GLUT4 detected by GFP tag (green). Individual antibody staining is shown in black and white, while the merged image shows all four colors with overlaps in white. Scale bars: 25 µm. (D and E) Representative IF staining for GLUT4 (green), CHC22 or sortilin (red) and p115 (blue) in HeLa-GLUT4 cells after treatment with siRNA targeting sortilin or IRAP or with non-targeting control. Scale bars: 5 µm. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Western Blot, Transfection, Molecular Weight, Immunofluorescence, Staining

    (A and B) Insulin-stimulated GLUT4 translocation in HeLa-GLUT4 cells as quantified by FACS analysis of surface:total GLUT4. Cells were pre-treated with siRNA targeting CHC22, CHC17, p115, GM130, sortilin, IRAP, sortilin plus IRAP, or with non-targeting control siRNA (siCon) as in , then incubated with (+) or without (-) insulin. For the experiments in (A), data is expressed as mean ± SEM, N=9, 10,000 cells acquired per experiment. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test **p<0.01, ***p<0.001, ****p<0.0001 versus untreated (-). For the experiments in (B), data is expressed as mean ± SEM, N=7, 10,000 cells acquired per experiment. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test *p<0.05, **p<0.01, ***p<0.001 versus untreated. (C) Proposed model for the roles of CHC22 in the human GLUT4 pathway. Newly synthesized GLUT4 traffics from the endoplasmic reticulum (ER) to the ER-to-Golgi Intermediate Compartment (ERGIC). At the ERGIC, a complex forms between IRAP and p115 that promotes binding of CHC22 clathrin and sequesters GLUT4 through its IRAP interaction (box A). Formation of the CHC22 clathrin coat at the ERGIC then facilitates sorting of GLUT4 to the intracellular region where GLUT4 storage vesicles (GSV) and insulin responsive GLUT4 vesicles (IRV) are formed. After insulin-mediated GLUT4 translocation and GLUT4 re-uptake (by CHC17 clathrin), a complex forms (box B) between endosomal GLUT4, sortilin and the clathrin adaptor GGA2, which promotes CHC22 recruitment. Endosomal GLUT4 sorting also involves clathrin adaptor AP1 ( ; ), further participating in CHC22 recruitment to endosomes. Formation of the CHC22 coat on sorting endosomes facilitates GLUT4 traffic to the TGN via the retrograde pathway, enabling replenishment of the intracellular GSV/IRV pool.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: (A and B) Insulin-stimulated GLUT4 translocation in HeLa-GLUT4 cells as quantified by FACS analysis of surface:total GLUT4. Cells were pre-treated with siRNA targeting CHC22, CHC17, p115, GM130, sortilin, IRAP, sortilin plus IRAP, or with non-targeting control siRNA (siCon) as in , then incubated with (+) or without (-) insulin. For the experiments in (A), data is expressed as mean ± SEM, N=9, 10,000 cells acquired per experiment. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test **p<0.01, ***p<0.001, ****p<0.0001 versus untreated (-). For the experiments in (B), data is expressed as mean ± SEM, N=7, 10,000 cells acquired per experiment. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test *p<0.05, **p<0.01, ***p<0.001 versus untreated. (C) Proposed model for the roles of CHC22 in the human GLUT4 pathway. Newly synthesized GLUT4 traffics from the endoplasmic reticulum (ER) to the ER-to-Golgi Intermediate Compartment (ERGIC). At the ERGIC, a complex forms between IRAP and p115 that promotes binding of CHC22 clathrin and sequesters GLUT4 through its IRAP interaction (box A). Formation of the CHC22 clathrin coat at the ERGIC then facilitates sorting of GLUT4 to the intracellular region where GLUT4 storage vesicles (GSV) and insulin responsive GLUT4 vesicles (IRV) are formed. After insulin-mediated GLUT4 translocation and GLUT4 re-uptake (by CHC17 clathrin), a complex forms (box B) between endosomal GLUT4, sortilin and the clathrin adaptor GGA2, which promotes CHC22 recruitment. Endosomal GLUT4 sorting also involves clathrin adaptor AP1 ( ; ), further participating in CHC22 recruitment to endosomes. Formation of the CHC22 coat on sorting endosomes facilitates GLUT4 traffic to the TGN via the retrograde pathway, enabling replenishment of the intracellular GSV/IRV pool.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Translocation Assay, Incubation, Synthesized, Binding Assay

    Quantification of alkaline phosphatase secretion index for HeLa-GLUT4 cells treated with siRNA targeting CHC22, CHC17, p115 or GM130 or with non-targeting control siRNA. The alkaline phosphatase secretion index is the ratio of secreted enzyme activity (culture medium) to total cellular activity (secreted plus cell lysate). Data expressed as mean ± SEM, N=13-19 independent samples across 2 independent assays. One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test **p<0.01 versus siControl.

    Journal: bioRxiv

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    doi: 10.1101/242941

    Figure Lengend Snippet: Quantification of alkaline phosphatase secretion index for HeLa-GLUT4 cells treated with siRNA targeting CHC22, CHC17, p115 or GM130 or with non-targeting control siRNA. The alkaline phosphatase secretion index is the ratio of secreted enzyme activity (culture medium) to total cellular activity (secreted plus cell lysate). Data expressed as mean ± SEM, N=13-19 independent samples across 2 independent assays. One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test **p<0.01 versus siControl.

    Article Snippet: The commercial anti-CHC22 from Proteintech was confirmed in our laboratory to be specific for CHC22 and not CHC17 ( ).

    Techniques: Activity Assay