Therefore, PnxIIIA appeared to tightly bind to proteins in the OM fraction. One candidate that interacts with PnxIIIA in the OM fraction is the gene product of pnxIIIE. Figure 4B shows the results of the Western blotting analysis of fractionated cells with anti-rPnxIIIE IgG. Signals appeared in the IM and OM fractions, and the estimated protein size was assumed to be the expected AZD1480 size of 30 kDa. These results may indicate that PnxIIIE exists mainly in the IM and OM fraction as a monomeric protein. Subsequently, we examined the in vitro interaction between rPnxIIIA and rPnxIIIE
by using a soluble protein cross-linker, BS3. The reaction mixture was then pulled down via immunoprecipitation (IP) by using anti-rPnxIIIA IgG. Figure 4C shows the results of the Western blotting analysis of cross-linking and the IP products detected with anti-rPnxIIIA IgG. The signal was detected at 250-kDa when only rPnxIIIA or rPnxIIIA and rPnxIIIE was used alone without cross-linking (Figure 4C, lane 1 and 3). However, the positions of their signals appeared higher than that of rPnxIIIA together with the parent selleck chemical 250-kDa rPnxIIIA when only rPnxIIIA or rPnxIIIA and rPnxIIIE was used after treatment with 50 mM BS3 (Figure 4C, lane 3 and 4). Furthermore, a shift of the signals
was observed with increasing reaction time when only rPnxIIIA was used after treatment with BS3 (Figure 4D). These results indicate that rPnxIIIA interacts learn more itself, and self-assembled oligomerized PnxIIIA is located in the OM Tobramycin fraction in P. pneumotropica ATCC 35149. Figure 4 Localization of PnxIIIA and the protein interaction analysis of rPnxIIIA. (A) Western blotting analysis of the cell fraction prepared
from P. pneumotropica ATCC 35149 cells and culture by using anti-rPnxIIIA IgG. Lanes: 1, SC fraction; 2, IM fraction; 3, OM fraction; 4, UC fraction. (B) Western blotting analysis of the cell fraction prepared from P. pneumotropica ATCC 35149 cells and culture by using anti-rPnxIIIE IgG. Lanes: 1, SC fraction; 2, IM fraction; 3, OM fraction; 4, UC fraction. (C) Western blotting analysis of rPnxIIIA by using anti-rPnxIIIA IgG after cross-linking with only rPnxIIIA or the rPnxIIIE protein and IP with anti-rPnxIIIA IgG. Lanes: 1, rPnxIIIA without cross-linking; 2, 20 μg of rPnxIIIA alone cross-linked with 50 mM BS3 for 60 min and immunoprecipitated; 3, mixture of both rPnxIIIA and rPnxIIIE proteins without cross-linking; 4, 20 μg of both rPnxIIIA and rPnxIIIE proteins cross-linked with 50 mM BS3 for 60 min and immunoprecipitated. (D) Western blotting analysis of rPnxIIIA by using anti-rPnxIIIA IgG after different treatment times with rPnxIIIA alone cross-linked with 50 mM BS3 and immunoprecipitated with anti-rPnxIIIA IgG.