Aberrant maturation of mutant perforin underlies the clinical diversity of hemophagocytic lymphohistiocytosis
J. Clin. Invest. Kimberly A. Risma, et al. 116:182 doi:10.1172/JCI26217 [Go to this article.]

Figure 5
Impaired maturation of perforin can be assayed in RBL-1 and RBL-2H3 cells. (A) Western blot analysis of perforin with missense mutations reveals 3 abnormal banding patterns. RBL-1 cells expressing WT or mutant perforin protein were lysed in 2% NP-40, and lysates were run on SDS-PAGE under nonreducing conditions and probed with polyclonal antibody P1-8. PRF1-N252S exhibits the typical WT pattern with 2 faint precursor bands (p1 and p2) and a more intense mature band (m) with greater mobility. PRF1-C73R, PRF1-A91V, and PRF1-R225W exhibit abnormal banding patterns as explained in the text. Although the same banding patterns are noted in RBL-2H3 cells expressing WT or mutant perforin, detection required 100 μg of protein. Detection was by P1-8 antibody. (B) Cytotoxic activity of human perforin bearing amino acid transitions of N252S, A91V, R225W, and C73R, expressed in RBL-2H3 cells. Perforin function was tested by its ability to lyse rbcs as described in Figure 3.