NPHP5 is polyubiquitinated in cells.

<p><b>A</b>) Flag-NPHP5 complexes from HEK293 cells were immuno-purified and subjected to mass spectrometric analysis. <b>B)</b> HEK293 cell lysates treated with or without a pan DUB inhibitor NEM were immunoprecipitated with anti-IgG (control) or anti-NPHP5 antibody and Western blotted with the indicated antibodies. IN, input. <b>C)</b> HEK293 cells were transfected with Flag or Flag-NPHP5 and HA or HA-Ub. Lysates were immunoprecipitated with anti-Flag antibody in 1% SDS and Western blotted with the indicated antibodies. IN, input. <b>D</b>) Flag-NPHP5 expressed in HEK293 cells was immuo-purified in the presence of NEM. Mass spectrometric quantitation of NPHP5 ubiquitination sites is presented. <b>E</b>) HEK293 cells were transfected with HA-Ub and Flag-NPHP5 wild type or hexamutant (K31RK33RK205RK263RK396RK528R). Lysates were immunoprecipitated with anti-Flag antibody in 1% SDS and Western blotted with the indicated antibodies. IN, input. <b>F)</b> Flag-NPHP5 expressed in HEK293 cells was immuno-purified in the presence of NEM. Mass spectrometric quantitation of Ub chain types attached to NPHP5 is presented. <b>G)</b> GFP-NPHP5 was co-expressed with Flag-Ub wild type, single mutant (K48R or K63R), double mutant (K48RK63R) or mutant in which all lysine residues were mutated to arginines (KallR) in HEK293 cells. Lysates were NEM treated and immunoprecipitated with anti-Flag antibody in 1% SDS and Western blotted with the indicated antibodies. IN, input. In <b>D, F)</b>, data were obtained from two independent experiments. Asterisks indicate non-specific bands.