FIG. 2.
Interaction of KPC2 with the proteasome. (A) Coelution of KPC2 with proteasome subunits on gel filtration chromatography. NIH 3T3 cells were incubated for 6 h in the presence of 10 μM MG132 or vehicle, lysed, and fractionated by gel filtration column chromatography. The resulting fractions (as well as cell lysates [lane L]) were subjected to immunoblot analysis with antibodies to KPC2, to S10 or to S12 subunits of the 19S proteasome complex, and to core α subunits (subunits 1, 2, 3, 5, 6, and 7) of the 20S proteasome. The elution positions of marker proteins (thyroglobulin, 669 kDa; ferritin, 440 kDa; bovine serum albumin, 67 kDa; RNase A, 13.7 kDa) and the void volume (Vo) are indicated. (B) Coimmunoprecipitation of the S5a subunit of the 19S proteasome complex with KPC2. HEK293T cells were incubated in the presence (left and right panels) or absence (right panel) of MG132 for 4 h, lysed, and subjected to immunoprecipitation (IP) with rabbit antibodies to KPC2 or with control rabbit IgG. The resulting precipitates were subjected to immunoblot analysis with antibodies to KPC1, KPC2, S5a, and glycogen synthase kinase-3β (internal control). A portion (1%) of the input lysates was also subjected directly to immunoblot analysis with the same antibodies. (C) Coimmunoprecipitation of S12 and KPC2. HEK293T cells were transfected with expression plasmids encoding KPC2 (tagged with the three-FLAG epitope) or S12 (tagged with the HA epitope), as indicated. They were then incubated with MG132 for 4 h, lysed, and subjected to immunoprecipitation with antibodies to FLAG or to HA. The resulting precipitates were subjected to immunoblot analysis with the same antibodies. A portion (1%) of the input lysates was also subjected directly to immunoblot analysis. (D) Coimmunoprecipitation of S10 with various mutants of KPC2. HEK293T cells expressing the indicated three-FLAG-tagged KPC2 derivatives or transfected with the empty vector (Mock) were incubated with MG132 for 4 h, lysed, and subjected to immunoprecipitation with antibodies to FLAG. The resulting precipitates were subjected to immunoblot analysis with antibodies to S10 or to FLAG. A portion (1%) of the input lysates was also subjected directly to immunoblot analysis with the same antibodies. IB, immunoblot; IP, immunoprecipitation.