Journal: International Journal of Molecular Sciences

Article Title: Usmani–Riazuddin Syndrome: Functional Characterization of a Novel c.196G>A Variant in the AP1G1 Gene and Phenotypic Insights Using Zebrafish as a Vertebrate Model

doi: 10.3390/ijms262110590

Figure Lengend Snippet: Subcellular localization of Golgi markers and AP1G1 in WT and c.196G>A mutant fibroblasts. ( A ) The cis-Golgi marker GM130 exhibited a typical compact perinuclear localization, consistent with an intact cis-Golgi structure. ( B ) The trans-Golgi network marker TGN46 exhibited a focused perinuclear pattern, outlining the organized architecture of the trans-Golgi cisternae. ( C ) AP1G1 was localized to the internal and peripheral regions of the cellular membrane, as well as in the perinuclear area (indicated by arrows), consistent with its role in vesicle formation in the trans-Golgi network and endosomes. ( D ) GM130 maintained a perinuclear distribution similar to that of WT, indicating that the overall organization of the cis-Golgi was preserved in the mutant cells. ( E ) TGN46 labeling appeared more dispersed and less defined, suggesting disorganization of the trans-Golgi network architecture in the presence of the mutation. ( F ). AP1G1 showed a punctate cytoplasmic distribution and was notably absent from membrane-associated regions, indicating defective membrane recruitment, likely resulting from impaired function of the mutant protein. In all panels, the nucleus is shown in blue via DAPI staining.

Article Snippet: After overnight incubation with blocking buffer (3% BSA, 1% glycine in PBS), the cells were subjected to immunolabeling using polyclonal anti TGN46, anti G130, anti AP1G1 (1:100; ABclonal, Düsseldorf, Germany) and goat anti-rabbit TRITC (1:50; Sigma Aldrich, Saint Louis, MO, USA) antibodies with fluorescein-conjugated goat anti-mouse IgG (Sigma-Aldrich) (1:50 in PBS, containing 3% BSA).

Techniques: Mutagenesis, Marker, Membrane, Labeling, Staining