FIGURE 4.

Dimerization of cochlins. HEK293T cells were cotransfected with HA- or FLAG-tagged WT, P53S, or G90E cochlin as indicated at a 1:1 ratio. Forty-eight hours after transfection (A–D), supernatants and lysates were collected. Western blotting (WB) of the culture supernatant (A and B) and cell lysates (C and D) was performed using anti-HA antibody after separation by 8% nonreducing SDS-PAGE (A and C, upper panels) or anti-CTF antibody after separation by 12% reducing SDS-PAGE (A and C, lower panels). The collected culture supernatant and lysates (B and D) were used to perform immunoprecipitation (IP) with anti-FLAG antibody-conjugated agarose beads. After washing, the FLAG immunocomplexes were eluted with FLAG peptide, and the eluates were analyzed by 8% nonreducing SDS-PAGE (B and D, upper panels) or 12% reducing SDS-PAGE (B and D, lower panels). The Western blots were probed with anti-HA antibody. The bands at ∼220 kDa in C (both upper and lower panels) are nonspecific. NS, nonspecific band. Cell lysates were collected 72 h after transfection, and 12% SDS-PAGE was conducted under reducing conditions (E). The Western blots were probed with anti-HA antibody. The results shown are representative of three independent experiments. DI-Coch, dimer cochlin; FL-Coch, full-length cochlin; OL-Coch, oligomer cochlin.