Rpgrip1l interacts with the 19S proteasomal subunit component Psmd2. (A) Coimmunoprecipitation experiments in NIH3T3 cells. Myc-tagged Psmd2 full-length protein and FLAG-tagged RID were transiently overexpressed and tested for interaction by coimmunoprecipitation from total cell lysates. Immunoprecipitation assays were performed by using an anti-FLAG antibody. Myc-Psmd2 coimmunoprecipitated with FLAG-RID (lane 3). Ctrl., control; IP, immunoprecipitation; WB, Western blot. (B) Myc-tagged Psmd2 full-length protein and FLAG/Strep/HA-tagged RID domain of Rpgrip1l were transiently overexpressed in HEK293T cells and tested for interaction by tandem affinity purification (TAP) tag experiments from total cell lysates. FLAG/Strep/HA-tagged RID was immunopurified using anti-FLAG beads, eluted with FLAG-protein, and recaptured on anti-Strep beads. The final eluation was performed by using biotin. After this sequential purification, we identified Myc-Psmd2 (lane 2) but not the unrelated protein Gatad1 (lane 6) in addition to purified FLAG/Strep/HA-RID. (C–E) Immunofluorescence on MEFs isolated from WT or Rpgrip1l^−/− E12.5 embryos (n = 3 embryos, respectively). (C) Centrosomes are marked by γ-tubulin as well as acetylated α-tubulin (α-Tub), and cell nuclei were marked by DAPI. Insets illustrate higher magnifications. (D) The ciliary axoneme and the BB are marked by acetylated α-tubulin and by γ-tubulin, respectively. The plot shows fluorescence intensities of the depicted representative image. (E) In situ proximity ligation assay (in situ PLA) on MEFs. Cell nuclei are marked by DAPI, and the ciliary axoneme are marked by transiently transfected Ift88-EYFP. Additional accumulation of Ift88-EYFP at the ciliary base is highlighted by yellow brackets. White arrowheads point to cilia. Bars: (C, all images; and D, overview) 10 µm; (D and E, magnifications) 1 µm.