Figure 1 | Blood Cancer Journal

Figure 1

From: Efficacy of an adapted granzyme B-based anti-CD30 cytolytic fusion protein against PI-9-positive classical Hodgkin lymphoma cells in a murine model

Figure 1

(a) Schematic structure of the protein EGb-Ki4(scFv). After purification, it is activated via enterokinase (‘E’)-digestion site indicated by an arrow. (b) Coomassie-stained SDS-PAGE gel with purified Gb-Ki4(scFv) before and after activation with enterokinase. (c) Results of apoptosis assay (annexin V/propidium iodide staining) on PI-9− L540cy cells and PI-9+ L428 cells after incubation with 33 nM Gb-Ki4(scFv). Numbers are % of cells within corresponding quadrant. (d and e) Expression of PI-9 in cHL cell lines L428, L1236 and L540cy. (d) Total soluble protein (40 μg/lane) was loaded on a gel for SDS-PAGE, and PI-9 was subsequently detected in a western blot using the antihuman PI-9 (clone 7D8) and GAM-PO. (e) 1 × 106 cells, as indicated, were fixed, permeabilized and stained with antihuman PI-9 (7D8) and GAM-FITC. PI-9 expression was determined by flow cytometry. (f) Complex formation of PI-9 and Gb-Ki4(scFv) within cell lysates of L540cy and L428 cells after pre-incubation with Gb-Ki4(scFv) ‘G’ or buffer ‘B’. Altogether, 40 μg total soluble protein was loaded on SDS-gel, and western blot was analyzed with antihuman GrB. Unspecific band was detected on the same height of CFP.

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