Figure 6. Cleaved Perforin-2 recovered after cellular infection with bacteria.
Perforin-2 −/− MEFs were transfected with human Perforin-2-GFP or GFP induced with murine IFN-γ and infected with the extracellular bacteria MRSA or EPEC. Following infection, bacteria were isolated, goat IgG was added to assess for nonspecific protein loss, and a portion filtered to distinguish bacteria from debris/soluble proteins. A noninfected control (first lane), demonstrates the selectivity of the differential centrifugations to remove mammalian cells. Figure 6—figure supplement 1 illustrates the recognition domain for human each Perforin-2 specific peptide generated antibody as well as verification. Fractions were probed against peptide-generated antibodies against the Perforin-2 domain (C93), and peptide generated antibodies against the MACPF domain (C186, C252). Commercial antibodies against ADA of EPEC and PBP of MRSA were utilized as bacterial markers. An additional band was observed following PBP immunoblot with a slightly higher molecular weight. This band was unspecific because it occurred in all samples including those derived from the experiments using EPEC. No signal was detected with previously validated peptide derived antibodies targeting the cytoplasmic domain of human Perforin-2 (C174), or peptide derived antibodies targeting a N-terminal portion of the Perforin-2 domain (C267) (Data not shown). In addition, commercial anti-human Perforin-2 antibody (detecting the cytoplasmic domain), clathrin, actin, and GFP also did not generate any signal (data not shown).
Figure 6—figure supplement 1. Human Perforin-2 peptide antibody validation.
