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. 2012 Jan 18;7(1):e29654. doi: 10.1371/journal.pone.0029654

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Zhang et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(a) MC was stable transfected with pIRES2-EGFP-OTUB1-myc. Then we performed IP with OTUB1 antibody and analyzed by SDS-PAGE using antibodies against the OTUB1, myc and DCN. (b) Reciprocal IP with anti-DCN antibody was analyzed using OTUB1 antibody. Lysate from MC (input) was used as a positive control, isotype matched nonspecific IgG as negative control. Lane 2(M) represents marker. (c) Cell lysates were subjected to immunoprecipitation with anti-DCN antibody followed by immunoblotting with an anti-ubiquitin (Ub) antibody. Lane 1 is normal mesangial cells (MC), cell lysates were analyzed by immunoblotting. Lane 2 is vacant vector pIRES2-EGFP transfected MC (MC/pIRES2) as control, Lane 3 is pIRES2-EGFP-OTUB1-myc transfected MC (MC/OTUB1), overexpression of Ub was seen in lane 3.