NPHP5 directly interacts with USP9X.

<p><b>A)</b> Flag or Flag-NPHP5 was expressed in HEK293 cells. Lysates were immunoprecipitated with anti-Flag antibody and Western blotted with the indicated antibodies. IN, input. <b>B)</b> (Top) IRCM1 and IRCM2 are two anti-NPHP5 antibodies raised against the C- and the N-terminus of NPHP5, respectively. Grey box, calmodulin-binding domain; white box, coiled-coil domain; black box; Cep290-binding domain. (Bottom) HEK293 lysates were immunoprecipitated with anti-Flag (control), anti-NPHP5 (IRCM1 or IRCM2) or anti-USP9X antibody and Western blotted with the indicated antibodies. IN, input. <b>C)</b> The ability of full-length NPHP5 (1-598(FL)) and various NPHP5 truncates to interact with USP9X is presented. <b>D)</b> Flag (control) or the indicated fragment of Flag-tagged NPHP5 was expressed in HEK293 cells. Lysates were immunoprecipitated with anti-Flag antibody and Western blotted with the indicated antibodies. IN, input. Asterisks indicate bands corresponding to the expected recombinant proteins. <b>E)</b> Purified GFP, GFP-USP9X wild type or catalytically inactive mutant (C1566S) bound to beads was mixed with purified Flag-NPHP5. Proteins recovered on beads were analyzed by Western blotting with the indicated antibodies. IN, input.