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. Author manuscript; available in PMC: 2011 Jan 12.

Published in final edited form as: Mol Cancer Res. 2010 Jan 12;8(1):119–130. doi: 10.1158/1541-7786.MCR-09-0277

A, sCLU interacts with COMMD1. Total proteins from PC-3, LN_mock and LN_sCLU were co-IP with anti-CLU or normal IgG followed by western blot using COMMD1 antibody. The inverse experiment was performed using COMMD1 antibody for co-IP. CLU, COMMD1 and vinculin were used for western blot (sCLU is detected as a 60-kDa glycosylated presecretory form of sCLU and is referred as psCLU in all western blot as proposed by Trougakos (43)). B, sCLU co-localizes with COMMD1 and ubiquitin. Immunofluorescence was performed in PC-3, LN_mock and LN_sCLU using CLU, COMMD1, and ubiquitin antibodies and DAPI for nuclei staining. C, sCLU associates with ubiquitin. Total proteins were incubated with ubiquitin-agarose matrix or Nickel agarose; and bound protein and input were used for western blots with CLU and Hsp90 antibodies. D, sCLU enhances ubiquitinated proteins. Total proteins (50ug) from LN_mock and LN_sCLU or PC-3 treated with sCLU or Scr siRNA were monitored for cleavage of the Suc-LLVY-AMC. Fluorescence was quantified using a spectrofluorometer (Fluoroskan Ascent FL, Thermo Labsystem). Data is expressed as mean ± SE. *, statistical significance (p<0.05) from 3 biological replicates.

A, sCLU interacts with COMMD1. Total proteins from PC-3, LN_mock and LN_sCLU were co-IP with anti-CLU or normal IgG followed by western blot using COMMD1 antibody. The inverse experiment was performed using COMMD1 antibody for co-IP. CLU, COMMD1 and vinculin were used for western blot (sCLU is detected as a 60-kDa glycosylated presecretory form of sCLU and is referred as psCLU in all western blot as proposed by Trougakos (43)). B, sCLU co-localizes with COMMD1 and ubiquitin. Immunofluorescence was performed in PC-3, LN_mock and LN_sCLU using CLU, COMMD1, and ubiquitin antibodies and DAPI for nuclei staining. C, sCLU associates with ubiquitin. Total proteins were incubated with ubiquitin-agarose matrix or Nickel agarose; and bound protein and input were used for western blots with CLU and Hsp90 antibodies. D, sCLU enhances ubiquitinated proteins. Total proteins (50ug) from LN_mock and LN_sCLU or PC-3 treated with sCLU or Scr siRNA were monitored for cleavage of the Suc-LLVY-AMC. Fluorescence was quantified using a spectrofluorometer (Fluoroskan Ascent FL, Thermo Labsystem). Data is expressed as mean ± SE. *, statistical significance (p<0.05) from 3 biological replicates.