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 8
Western blot analysis of RBL-1 expressing PRF1 missense mutations with absent mature band formation. (A) Mutant proteins demonstrated an absence of the mature band by nonreducing SDS-PAGE followed by Western blot using antibodies as labeled. WT perforin is shown for comparison. The use of 2 different antibodies (H315 and P1-8) was critical to demonstrate the presence of the 70-kDa band under reducing conditions for PRF1-F157V and PRF1-R225W. Fifty micrograms protein was loaded per lane. (B) RBL-1 cells expressing WT, V50M, and R225W perforin were disrupted and fractionated through Percoll gradients. Samples from every second fraction of the gradients were then analyzed for the presence of precursor and mature forms of perforin on nonreducing SDS-PAGE. WT perforin is shown for comparison. “C” indicates a cell lysate from RBL-1 cells expressing WT human perforin (4 μg protein). For PRF1-V50M, 4 μg protein was loaded per lane; 1.5 μg was loaded for WT PRF1 and PRF1-R225W. Detection was by P1-8 antibody.