Journal:
Article Title: Localization of ADP-ribosylation factor domain protein 1 (ARD1) in lysosomes and Golgi apparatus
doi:
Figure Lengend Snippet: Subcellular localization of ARD1. (A) Subcellular fractionation of cells overexpressing ARD1. Five 100-mm dishes of HeLa cells were transfected with pcDNA(ARD1). After 48 h, cells were homogenized in 2 ml of buffer A (see legend for Fig. Fig.1)1) and crude membranes were obtained by centrifugation at 100,000 × g for 30 min as described in Fig. Fig.1.1. Membranes were layered on a continuous sucrose density gradient [1–2.2 M sucrose and 10 mM Tris (pH 7.4)] as described (17) and centrifuged for 1 h at 100,000 × g. Twelve 1-ml fractions were collected from the top and diluted in 20 mM Tris (pH 8.0) with 1 mM EDTA; membranes were collected by centrifugation (30 min at 100,000 × g) of each fraction and protein was assayed according to the Bradford procedure (16). The distribution of membranes on the gradient has been described (14, 17). Samples of proteins (40 μg) from each fraction were subjected to SDS/PAGE, transferred to nitrocellulose membranes, and reacted with antibodies against Nt-ARD1 (1:10,000), βCOP (1:200), or LAMP1 (1:1,000). Positions of protein standards (kDa) are on the left. (B) Intracellular distribution of ARD1 by immunofluorescence. COS 7 cells transfected with pcDNA3.1(ARD1) were incubated with 4 ml of warm (37°C) DMEM (a) or DMEM containing brefeldin A, 10 μg/ml (b) for 10 min, then incubated simultaneously with the rabbit anti-Nt-ARD1 (1:10,000) and the mouse anti-p58 (1:100) antibodies, followed by anti-rabbit IgG–rhodamine and anti-mouse IgG–fluorescein isothiocyanate antibodies. Transfected HeLa cells were incubated with the rabbit anti-Nt-ARD1 (1:15,000) (c–d) and the mouse anti-LAMP1 (1:1,000) (c) followed by secondary antibodies. Some cells were incubated for 1 h with 60 nM Lysotracker (lysosomal marker) in DMEM before fixation and permeabilization with digitonin (d). Transfected NIH 3T3 cells were incubated at 37°C for 15 min in AcR solution (19) (e) or nocodazole, 5 μg/ml, for 60 min (f) before immunostaining. Results have been repeated at least once with different cell preparations.
Article Snippet: Antibodies against βCOP, p58, and adaptor protein 1 (AP1) were purchased from Sigma, as were the secondary antibodies used for Western blotting and immunofluorescence studies.
Techniques: Fractionation, Transfection, Centrifugation, SDS Page, Immunofluorescence, Incubation, Marker, Immunostaining
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![Subcellular localization of ARD1. (A) Subcellular fractionation of cells overexpressing ARD1. Five 100-mm dishes of HeLa cells were transfected with pcDNA(ARD1). After 48 h, cells were homogenized in 2 ml of buffer A (see legend for Fig. Fig.1)1) and crude membranes were obtained by centrifugation at 100,000 × g for 30 min as described in Fig. Fig.1.1. Membranes were layered on a continuous sucrose density gradient [1–2.2 M sucrose and 10 mM Tris (pH 7.4)] as described (17) and centrifuged for 1 h at 100,000 × g. Twelve 1-ml fractions were collected from the top and diluted in 20 mM Tris (pH 8.0) with 1 mM EDTA; membranes were collected by centrifugation (30 min at 100,000 × g) of each fraction and protein was assayed according to the Bradford procedure (16). The distribution of membranes on the gradient has been described (14, 17). Samples of proteins (40 μg) from each fraction were subjected to SDS/PAGE, transferred to nitrocellulose membranes, and reacted with antibodies against Nt-ARD1 (1:10,000), βCOP (1:200), or LAMP1 (1:1,000). Positions of protein standards (kDa) are on the left. (B) Intracellular distribution of ARD1 by immunofluorescence. COS 7 cells transfected with pcDNA3.1(ARD1) were incubated with 4 ml of warm (37°C) DMEM (a) or DMEM containing brefeldin A, 10 μg/ml (b) for 10 min, then incubated simultaneously with the rabbit anti-Nt-ARD1 (1:10,000) and the mouse <t>anti-p58</t> (1:100) antibodies, followed by anti-rabbit IgG–rhodamine and anti-mouse IgG–fluorescein isothiocyanate antibodies. Transfected HeLa cells were incubated with the rabbit anti-Nt-ARD1 (1:15,000) (c–d) and the mouse anti-LAMP1 (1:1,000) (c) followed by secondary antibodies. Some cells were incubated for 1 h with 60 nM Lysotracker (lysosomal marker) in DMEM before fixation and permeabilization with digitonin (d). Transfected NIH 3T3 cells were incubated at 37°C for 15 min in AcR solution (19) (e) or nocodazole, 5 μg/ml, for 60 min (f) before immunostaining. Results have been repeated at least once with different cell preparations.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1124/pmc00021124/pmc00021124__pq1481736003.jpg)
