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. 2010 Oct;24(10):4058–4067. doi: 10.1096/fj.10-162925

Figure 2.

Figure 2.

Regions required for PKD1L3 and PKD2L1 to interact. Coimmunoprecipitation assays were performed using PKD1L3, PKD2L1, and their deletion mutants. A) Control Western blot analysis indicating expression of PKD1L3, PKD2L1, and their deletion mutants. i) C-terminally truncated PKD1L3 mutants and full-length PKD2L1. ii) PKD1L3 deletion mutants and full-length PKD2L1. iii) PKD1L3TM6-CT and C- or N-terminally truncated PKD2L1 mutants. iv) Full-length PKD2L1 and PKD2L1ΔCT. B) i) Interaction between C-terminally truncated PKD1L3 mutants and PKD2L1. When HA-tagged PKD1L3 or C-terminally truncated PKD1L3 mutants were precipitated, FLAG-tagged PKD2L1 proteins coprecipitated (lanes 1–3). Less FLAG-tagged PKD2L1 protein coprecipitated with the PKD1L3NT-TM6 protein (lane 4). We could not detect any signals for HA-tagged PKD1L3FL in either the lysis (A) or the immunoprecipitation, likely because of low transfer efficiency of high-molecular-mass protein (∼234 kDa) from gels to PVDF membranes. Immunostaining analysis under permeabilized conditions demonstrated that protein expression levels were comparable among PKD1L3FL, PKD1L3NT-TM11, PKD1L3NT-TM10, and PKD1L3NT-TM6 (Fig. 3). ii) Region of PKD1L3 that is required for interaction with PKD2L1. When HA-tagged PKD1L3 deletion mutants were precipitated, FLAG-tagged PKD2L1 proteins were coprecipitated (lanes 5–10). Little FLAG-tagged PKD2L1 protein coprecipitated with the PKD1L3TM11-CT protein (lane 11). iii) Region of PKD2L1 that is required for heteromeric interaction with PKD1L3. When HA-tagged PKD1L3TM6-CT was precipitated, FLAG-tagged PKD2L1 N-terminal or C-terminal truncated proteins coprecipitated (lanes 12–14, 16); in contrast, little PKD2L1CT protein coprecipitated (lane 15). iv) Region of PKD2L1 that is required for homomeric interaction. When HA-tagged PKD2L1 or PKD2L1ΔCT was precipitated, FLAG-tagged PKD2L1 or PKD2L1ΔCT protein coprecipitated (lanes 17–19). Solid arrowheads indicate PKD1L3 deletion mutants; open arrowheads indicate PKD2L1 or PKD2L1 deletion mutants; brackets indicate high-molecular-mass oligomers. Data are representative of ≥3 independent experiments.