ergic53 cl b9 Search Results


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IER3IP1 is a protein of the early secretory pathway. a) Model of IER3IP1 structure. Numbers indicate first and last amino acids of the two predicted transmembrane domains ( https://alphafold.ebi.ac.uk/entry/Q9Y5U9 ). The localization of the pathogenic mutations is indicated (red asterisks). b) IER3IP1 KO and re-expression of IER3IP1 cell line variants were analyzed by WB using anti-IER3IP1. Lysates were assessed for expression of the indicated proteins, with β-actin serving as loading control (n = 3 independent experiments). c, d) Indicated cells were fixed, co-labeled with anti-IER3IP1 (red) and (c) Sec31 (green) or (d) <t>ERGIC53</t> (green), and Hoechst nuclear dye (blue), and analyzed by fluorescence microscopy. Arrows indicate overlap of IER3IP1 with the respective marker. e) The numbers of Sec31 objects per cell in cells processed as in (c) were analyzed using Cell Profiler. Values obtained from individual images are represented as small circles and mean values for independent experiments as filled circles (n = 3 independent experiments, mean ± SD, One-way Anova with Dunnett’s post-hoc test, n Control = 506, n KO1 = 534, n KO1+WT = 644, n KO1+P.L78P = 493 cells). f) Cells were transfected with EGFP-Sar1b (H79G), fixed after 24 h, labeled with anti-IER3IP1 (blue) and Sec31A (red), and analyzed by fluorescence microscopy. Arrows indicate co-localization of the three proteins. Scale bars, 10 μm
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IER3IP1 is a protein of the early secretory pathway. a) Model of IER3IP1 structure. Numbers indicate first and last amino acids of the two predicted transmembrane domains ( https://alphafold.ebi.ac.uk/entry/Q9Y5U9 ). The localization of the pathogenic mutations is indicated (red asterisks). b) IER3IP1 KO and re-expression of IER3IP1 cell line variants were analyzed by WB using anti-IER3IP1. Lysates were assessed for expression of the indicated proteins, with β-actin serving as loading control (n = 3 independent experiments). c, d) Indicated cells were fixed, co-labeled with anti-IER3IP1 (red) and (c) Sec31 (green) or (d) ERGIC53 (green), and Hoechst nuclear dye (blue), and analyzed by fluorescence microscopy. Arrows indicate overlap of IER3IP1 with the respective marker. e) The numbers of Sec31 objects per cell in cells processed as in (c) were analyzed using Cell Profiler. Values obtained from individual images are represented as small circles and mean values for independent experiments as filled circles (n = 3 independent experiments, mean ± SD, One-way Anova with Dunnett’s post-hoc test, n Control = 506, n KO1 = 534, n KO1+WT = 644, n KO1+P.L78P = 493 cells). f) Cells were transfected with EGFP-Sar1b (H79G), fixed after 24 h, labeled with anti-IER3IP1 (blue) and Sec31A (red), and analyzed by fluorescence microscopy. Arrows indicate co-localization of the three proteins. Scale bars, 10 μm

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: IER3IP1-mutations cause microcephaly by selective inhibition of ER-Golgi transport

doi: 10.1007/s00018-024-05386-x

Figure Lengend Snippet: IER3IP1 is a protein of the early secretory pathway. a) Model of IER3IP1 structure. Numbers indicate first and last amino acids of the two predicted transmembrane domains ( https://alphafold.ebi.ac.uk/entry/Q9Y5U9 ). The localization of the pathogenic mutations is indicated (red asterisks). b) IER3IP1 KO and re-expression of IER3IP1 cell line variants were analyzed by WB using anti-IER3IP1. Lysates were assessed for expression of the indicated proteins, with β-actin serving as loading control (n = 3 independent experiments). c, d) Indicated cells were fixed, co-labeled with anti-IER3IP1 (red) and (c) Sec31 (green) or (d) ERGIC53 (green), and Hoechst nuclear dye (blue), and analyzed by fluorescence microscopy. Arrows indicate overlap of IER3IP1 with the respective marker. e) The numbers of Sec31 objects per cell in cells processed as in (c) were analyzed using Cell Profiler. Values obtained from individual images are represented as small circles and mean values for independent experiments as filled circles (n = 3 independent experiments, mean ± SD, One-way Anova with Dunnett’s post-hoc test, n Control = 506, n KO1 = 534, n KO1+WT = 644, n KO1+P.L78P = 493 cells). f) Cells were transfected with EGFP-Sar1b (H79G), fixed after 24 h, labeled with anti-IER3IP1 (blue) and Sec31A (red), and analyzed by fluorescence microscopy. Arrows indicate co-localization of the three proteins. Scale bars, 10 μm

Article Snippet: ERGIC53 cl B9 , sc-271517, Santa Cruz Biotechnology , IF 1:200.

Techniques: Expressing, Control, Labeling, Fluorescence, Microscopy, Marker, Transfection

Cargo exit from the ER is delayed in IER3IP1 KO cells and in cells expressing pathogenic mutations. a) Cells fixed and co-labeled with anti-ERGIC53 (green), anti-Sec16A (red) and Hoechst (blue) were analyzed by fluorescence microscopy. ERGIC53 (b) or Sec16A (c) objects were counted in the cell periphery (small arrows) or in the perinuclear region (large arrows) using Cell Profiler. The areas of the perinuclear regions are delineated in Suppl. Fig. a. Ratios of the mean number of peripheral and perinuclear objects are shown for each condition (mean ± SD, unpaired two-tailed t-test, > 222 cells/condition). b) n = 4 except for n KO1+p.V21G = 3. c) n = 3. d) HeLa cells were transfected with the indicated constructs. IER3IP1 and EGFP-ERGIC53 were immunoprecipitated from the lysates using anti-IER3IP1 or anti-EGFP antibodies, and then analyzed by immunoblotting (n = 5 independent experiments). e–g) Indicated cell lines were transfected with Str-STIM-ST-SBP-mCherry (ST-mCherry). After 24 h, cells were incubated with biotin for 17 min, fixed and co-stained with anti-GM130 (Golgi marker, green) and Hoechst dye (blue), and analyzed by fluorescence microscopy. f) The areas of GM130-stained Golgi measured with Fiji in individual cells (small circles), and the color-coded mean values per experiment (large, filled circles) are shown (n = 4 independent experiments, mean ± SD, One-way Anova with Dunnett’s post-hoc test; n Control = 476, n KO1 = 435, n KO1+WT = 418, n KO1+p.L78P = 550, n KO1+p.V21G = 252, n KO1+p.T79∆ = 312 cells). g) Ratios between Golgi and total (entire cell) ST-mCherry mean fluorescence intensities, measured using Fiji, are shown for individual cells (small circles), and for independent experiments (mean values indicated as large, filled circles) (n = 5 independent experiments, mean ± SD, One-way Anova with Dunnett’s post-hoc test; n Control = 515, n KO1 = 475, n KO1+WT = 476, n KO1+p.L78P = 624, n KO1+p.V21G = 293, n KO1+p.T79∆ = 368 cells)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: IER3IP1-mutations cause microcephaly by selective inhibition of ER-Golgi transport

doi: 10.1007/s00018-024-05386-x

Figure Lengend Snippet: Cargo exit from the ER is delayed in IER3IP1 KO cells and in cells expressing pathogenic mutations. a) Cells fixed and co-labeled with anti-ERGIC53 (green), anti-Sec16A (red) and Hoechst (blue) were analyzed by fluorescence microscopy. ERGIC53 (b) or Sec16A (c) objects were counted in the cell periphery (small arrows) or in the perinuclear region (large arrows) using Cell Profiler. The areas of the perinuclear regions are delineated in Suppl. Fig. a. Ratios of the mean number of peripheral and perinuclear objects are shown for each condition (mean ± SD, unpaired two-tailed t-test, > 222 cells/condition). b) n = 4 except for n KO1+p.V21G = 3. c) n = 3. d) HeLa cells were transfected with the indicated constructs. IER3IP1 and EGFP-ERGIC53 were immunoprecipitated from the lysates using anti-IER3IP1 or anti-EGFP antibodies, and then analyzed by immunoblotting (n = 5 independent experiments). e–g) Indicated cell lines were transfected with Str-STIM-ST-SBP-mCherry (ST-mCherry). After 24 h, cells were incubated with biotin for 17 min, fixed and co-stained with anti-GM130 (Golgi marker, green) and Hoechst dye (blue), and analyzed by fluorescence microscopy. f) The areas of GM130-stained Golgi measured with Fiji in individual cells (small circles), and the color-coded mean values per experiment (large, filled circles) are shown (n = 4 independent experiments, mean ± SD, One-way Anova with Dunnett’s post-hoc test; n Control = 476, n KO1 = 435, n KO1+WT = 418, n KO1+p.L78P = 550, n KO1+p.V21G = 252, n KO1+p.T79∆ = 312 cells). g) Ratios between Golgi and total (entire cell) ST-mCherry mean fluorescence intensities, measured using Fiji, are shown for individual cells (small circles), and for independent experiments (mean values indicated as large, filled circles) (n = 5 independent experiments, mean ± SD, One-way Anova with Dunnett’s post-hoc test; n Control = 515, n KO1 = 475, n KO1+WT = 476, n KO1+p.L78P = 624, n KO1+p.V21G = 293, n KO1+p.T79∆ = 368 cells)

Article Snippet: ERGIC53 cl B9 , sc-271517, Santa Cruz Biotechnology , IF 1:200.

Techniques: Expressing, Labeling, Fluorescence, Microscopy, Two Tailed Test, Transfection, Construct, Immunoprecipitation, Western Blot, Incubation, Staining, Marker, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: IER3IP1-mutations cause microcephaly by selective inhibition of ER-Golgi transport

doi: 10.1007/s00018-024-05386-x

Figure Lengend Snippet:

Article Snippet: ERGIC53 cl B9 , sc-271517, Santa Cruz Biotechnology , IF 1:200.

Techniques: Western Blot, Immunofluorescence, Immunoprecipitation, Recombinant

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: IER3IP1-mutations cause microcephaly by selective inhibition of ER-Golgi transport

doi: 10.1007/s00018-024-05386-x

Figure Lengend Snippet:

Article Snippet: ERGIC53 cl B9 , sc-271517, Santa Cruz Biotechnology , IF 1:200.

Techniques: