Figure 2.

ICA69 and PICK1 form tight complexes in vitro and in vivo. A, Myc-ICA69 was transfected into 293T cells with or without GFP-PICK1. PICK1 was immunoprecipitated using an anti-GFP antibody. Anti-GFP antibody (top) or anti-myc antibody (bottom) was used for Western blotting. Expression of GFP-PICK1 and myc-ICA69 in 293T cells is indicated in the INPUTs, which are the cell lysates used for IP. The IP products indicate that myc-ICA69 was only pulled down in the presence of GFP-PICK1 but not in the control, when myc-ICA69 was expressed alone. B, GFP-ICA69 and myc-PICK1 were transfected into 293T cells and immunoprecipitation was performed as described in A. Similarly, myc-PICK1 was only pulled down when ICA69 was present. C, Coimmunoprecipitation from rat brain homogenate was performed using an anti-PICK1 antibody. The antibody pulled down PICK1 itself (top) and a significant amount of ICA69 (bottom), indicating that ICA69 and PICK1 interact with each other in vivo. In the control experiments, when PICK1 antibody was preblocked by the antigenic peptide, or when preimmune serum or unrelated anti-GFP serum was used, both PICK1 and ICA69 were not precipitated. D, PICK1 was consecutively immunoprecipitated with anti-PICK1 antibodies from rat brain homogenate. The rat brain homogenate before immunoprecipitation was designated as INPUT. The rat brain homogenate after the first immunoprecipitation was designated as AIP1. AIP1 was subsequently immunoprecipitated with PICK1 antibodies again, and the rat brain homogenate after the second immunoprecipitation was designated as AIP2. Immunoprecipitation was performed consecutively until almost all of the PICK1 was depleted from the rat brain homogenate, which usually took two to three rounds of immunoprecipitation. Equal volumes of INPUT and AIP samples were loaded, the intensity of INPUT was normalized to 1, and the intensities of the AIPs were normalized in relation to corresponding INPUT. The last AIP from the consecutive immunoprecipitation was used for the quantification and bar chart. E, The quantification data show that when PICK1 is almost completely depleted (7.4 ± 3.2% in AIP, mean ± SEM, n = 3), there is ∼16.8 ± 7.6% of ICA69 left in AIP. This translates to ∼90% of ICA69 being immunoprecipitated if PICK1 was 100% immunoprecipitated. F, Consecutive coimmunoprecipitation was performed as described in D using anti-ICA69 antibodies. G, The quantification data show that when ICA69 was nearly completely depleted (5.6 ± 3.6% in AIP, mean ± SEM, n = 3), there is ∼28.2 ± 12.2% of PICK1 left in AIP. This translates to 76% of PICK1 associating with ICA69 in rat brain homogenates.