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. 2012 Jul 31;31(17):3607–3619. doi: 10.1038/emboj.2012.212

Figure 6.

Figure 6

Determination of the Staphopain A cleavage site in CXCR2. (A) Staphopain A cleaves the CXCR21–48-GB1-His protein. Different concentrations of Staphopain A were incubated with 6.75 μM CXCR21–48-GB1-His protein for 15 min at 37°C. Samples were analysed by SDS–PAGE and Instant blue staining. Lane 6: CXCR21–48-GB1-His plus 150 nM Staphopain A plus 1 μM Staphostatin A; lane 7: Staphostatin A alone. (B) N-terminal sequencing of the CXCR2 cleavage product generated by incubation of 6.75 μM CXCR21–48-GB1-His protein with 150 nM Staphopain A for 15 min at 37°C. Sequencing was performed once. (C) Staphopain A cleavage of CXCR21–48-GB1-His (left, LLDA) or CXCR21–48-GB1-His mutated with a Proline at position 34 (right, LPDA). CXCR2 protein at 6.75 μM, 15 min at 37°C; proteins were stained using Instant Blue. (D) Staphopain A cleaves CXCR2 in the presence of plasma. CXCR21–48-GB1-His (1.5 μM) was incubated with Staphopain A with or without 10% human plasma for 30 min at 37°C. CXCR2 was visualized by immunoblotting using anti-CXCR2 (N-terminus specific) antibodies. (A, C and D) Representatives of three separate experiments are shown.