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. Author manuscript; available in PMC: 2015 Jan 13.
Published in final edited form as: J Biol Chem. 2006 Apr 6;281(23):16108–16116. doi: 10.1074/jbc.M600760200

FIGURE 5. PACS-2 and COPI localize H69K:fur/f/ha to the ER.

FIGURE 5

A, A7 cells expressing fur/f/ha or H69K:fur/f/ha were metabolically labeled with 32Pi, lysed, immunoprecipitated (IP) with mAb M2, separated by SDS-PAGE, and analyzed by autoradiography (upper panel) and Western blot (WB) using anti-furin PA1-062 (lower panel). B, glutathione S-transferase or GST-FurinCD, which contains the 56-amino acid furin cytosolic domain, was preincubated or not with CK2 and then incubated with thioredoxin-tagged PACS-2 FBR, which encodes the cargo binding region of PACS-2 (17, 25), separated by SDS-PAGE, and analyzed by Western blot using anti-Trx mAb. C, A7 cells were treated with control (scr.) or siRNAs specific for PACS-2 or β-COP for 48 h, lysed, separated by SDS-PAGE, and analyzed by Western blot using anti-PACS-2 and anti-β-COP antibodies. The blots were also incubated with anti-tubulin mAb to control for protein loading. D, A7 cells were treated with the indicated siRNAs for 48 h and then infected with virus expressing H69K:fur/f/ha for an additional 4 h. The cells were then processed for immunofluorescence microscopy and stained with mAb HA.11 and anti-PDI followed by subtype-specific fluorescently labeled secondary antibodies.