Figure 5.
Mutant COPZ1 caused deregulated protein expression, phosphorylation, and retrograde Golgi-to-ER trafficking. (A-C) Protein expression levels in COPZ1 mutant samples relative to their corresponding AAVS1-edited controls, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein loading control levels, as assessed by DigiWest assay. The green dotted line indicates the expression level in COPZ1 WT samples (n = 2). (D) Immunofluorescence images of the CtxB transport assay in AAVS1-edited control and COPZ1-TR adult human primary dermal fibroblasts (HDFa), costained for GM130 (Golgi marker, red), SERCA2 (endoplasmic reticulum calcium pump, green), and nucleus (DAPI, blue). CtxB (cholera toxin subunitB, purple) was analyzed at 2 hours (time 0) and 10 hours (time 8 hours) postexposure to assess intracellular trafficking. The analysis was conducted at 2 hours (time 0) and 10 hours (time 8 hours) after CtxB exposure. Scale bars represent 10 μm. (E) Graphs depict the quantifications of the degree of CtxB colocalization with the Golgi (left) and the ER (right). Data are presented as means ± SD from 3 independent experiments. Statistical significance, ∗P < .05; ∗∗P < .01; ER, endoplasmic reticulum; ns, not significant.

Mutant COPZ1 caused deregulated protein expression, phosphorylation, and retrograde Golgi-to-ER trafficking. (A-C) Protein expression levels in COPZ1 mutant samples relative to their corresponding AAVS1-edited controls, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein loading control levels, as assessed by DigiWest assay. The green dotted line indicates the expression level in COPZ1 WT samples (n = 2). (D) Immunofluorescence images of the CtxB transport assay in AAVS1-edited control and COPZ1-TR adult human primary dermal fibroblasts (HDFa), costained for GM130 (Golgi marker, red), SERCA2 (endoplasmic reticulum calcium pump, green), and nucleus (DAPI, blue). CtxB (cholera toxin subunitB, purple) was analyzed at 2 hours (time 0) and 10 hours (time 8 hours) postexposure to assess intracellular trafficking. The analysis was conducted at 2 hours (time 0) and 10 hours (time 8 hours) after CtxB exposure. Scale bars represent 10 μm. (E) Graphs depict the quantifications of the degree of CtxB colocalization with the Golgi (left) and the ER (right). Data are presented as means ± SD from 3 independent experiments. Statistical significance, ∗P < .05; ∗∗P < .01; ER, endoplasmic reticulum; ns, not significant.

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