FIG 3.
PA2066 and PA0256 are putative Hcp cargoes. (A) Solubility test for PA2066; E. coli BL21 cells expressed either HA-tagged PA2066 alone or with Hcp2-His. After induction, cells were grown at 18°C overnight, harvested, and resuspended in purification resuspension buffer, sonicated, and centrifuged. Western blot of postexpression, insoluble, and soluble samples. (B) Western blot of Hcp2-His and PA2066-HA copurification; lanes labeled at the top are postexpression sample, insoluble and soluble samples after sonication and clarification, flowthrough (FT) from the Ni-NTA column, wash fraction before elution, Ni fractions corresponding to the elution peak, and SEC fractions corresponding to the gel filtration peak. The Hcp2-His with PA2066-HA copurification Western blot membrane was cut so that the wash-SEC fractions could be exposed longer to check that the wash fraction was clear and if there were bands detectable in the SEC peak fractions with anti-HA antibody. (C) SEC chromatograph of purified Hcp2-His and PA2066-HA. Hcp3-His and HA-PA0256 copurification Western blot (D), SEC chromatograph (E), and Coomassie blue stain (F). (G) Copurification of Hcp2T71Q-His and PA2066-HA. (H) Copurification of Hcp3T60Q-His and HA-PA0256. The antibodies used are labeled on the right: top, anti-HA antibody (BioLegend) at 1:1,000 concentration; bottom, either anti-Hcp2 antibody (Eurogentec) at 1:500 concentration or anti-His antibody (GenScript) at 1:1,000 concentration. Molecular weight standards are on the left.