AZI1 interacts with the BBSome through BBS4.

<p><b>A</b>) A silver stained gel from GFP IP sample of testis lysate. Lane 1 shows the wild-type (WT) precipitate and lane 2 is the precipitate of testis lysate from transgenic (TG) animals. The band labeled with an asterisk is the 120 KDa band identified as Azi1 by mass spectrometry. <b>B</b>) Western blot showing interaction of BBS4 with AZI1 and PCM1. FLAG and S tagged BBSome subunits were transfected in 293T cells and Co-IP was performed using FLAG agarose beads. The top blot shows efficient transfection of the BBS subunits detected by anti-FLAG antibody. The lower two blots were immunoblotted with AZI1 and PCM1 antibody, respectively. <b>C</b>) FLAG-BBS4 stable cell (293T) was co-immunoprecipitated with FLAG agarose beads. Input, supernatant and eluate were immunoblotted with, antibody against GAPDH to show that equal amounts were loaded, antibody against FLAG to show efficient precipitation of BBS4 by FLAG beads, and antibody against AZI1 to show amounts of AZI1 precipitated by BBS4 in our cell line. <b>D</b>) 293T cells stably expressing the FLAG tagged BBSome subunits BBS4, 5, 8, and 9 were co-IPed with FLAG agarose beads and immunoblotted with AZI1 antibody. Although all the subunits were able to precipitate AZI1, BBS4 showed the most efficient pull-down. <b>E</b>) Co-IP using AZI1 antibody was performed in 293T cells followed by Western blotting with different BBS antibody to show interaction of endogenous BBS proteins and AZI1.