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. 2011 Jan 21;286(13):11242–11253. doi: 10.1074/jbc.M110.171728

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 8. — Co-localization and co-immunoprecipitation of cyclophilin B with NaDC1. A, subcellular localization of CypB and NaDC1 in non-transfected HEK293 cells treated ± 200 ng/ml brefeldin A, Western analyses of the last seven fractions out of 10 (numbered 1–7), in which GM130 (cis-Golgi Matrix protein 130; cis-Golgi marker) and PDI (ER marker) were immunodetected. In non-treated cells, CypB co-localized with immature NaDC1 in the ER (fractions 6 and 7). In brefeldin A-treated cells, the expression of the immature NaDC1 form and CypB was increased in the ER (fractions 6 and 7). B, co-immunoprecipitation of CypB with NaDC1: HA-tagged CypB was co-injected with either FLAG-tagged NaDC1 or wt TRPV6 (positive control). Non-injected oocytes were used as negative control. NaDC1 and TRPV6 were immunoprecipitated (IP) with rabbit polyclonal anti-FLAG and chicken polyclonal anti-huTRPV6 antibodies, respectively. Western membranes were immunoblotted (IB) with the mouse monoclononal anti-HA. A CypB immunoreactive band (∼20 kDa) was detected in all co-immunoprecipitation studies.