Fig. 3. Self-aggregation of PCM-1. (A) Yeast two-hybrid analyses. The cDNA encoding aa 1-484 or aa 745-1273 of XPCM-1 (p53 as a control) was fused to the DNA-binding domain (DNA-BD) in the pLexA vector and the cDNA encoding aa 1-711 or aa 745-1273 (SV40 large T-antigen as a control) to the activation domain (AD) in the pB42AD vector. DNA-BD and AD constructs were then transformed into yeast. Notice that aa 1-484 and aa 745-1273 bound directly to aa 1-711 and aa 745-1273, respectively. (B) Overexpression of a GST fusion protein with XPCM-1 fragment (aa 745-1271) in insect Sf9 cells. As seen in A6 cells (Fig. 2B), electron-dense large aggregates were formed within the cytoplasm abundantly (a). These Sf9 cells were lysed with 1% Triton X-100 and the lysate was separated by centrifugation. The aggregates were partially isolated as a pellet (b). Bars, 1 µm. (C) Components of the partially isolated aggregates. The pellet (ppt) and supernatant (sup) were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). The pellet contained only one major band, around 83 kDa (sometimes divided into two bands for an unknown reason), which was identified as the GST-XPCM-1 fragment by immunoblotting with anti-GST mAb (western). Bars represent molecular masses of 203, 116 and 83 kDa from the top. (D) In vitro binding assay. A column consisting of GST fusion proteins with a XPCM-1 fragment (aa 745-1271 and aa 1020-1271) was constructed, to which the whole lysate of Xenopus interphase egg extract was applied. Bound proteins were then eluted (CBB) and immunoblotted with anti-XPCM-1 pAb (western). Arrows indicate the GST fusion proteins. Notice that endogenous full-length XPCM-1 bound to aa 745-1271 but not to aa 1020-1271. Bars represent molecular masses of 203, 116 and 83 kDa from the top.