![Fig. 7. Engagement of PSGL-1 by the monoclonal antibody PL2 triggers genistein-sensitive, β2-integrin–dependent PMN aggregation and stimulates protein-tyrosine phosphorylation. (A) PMN were preincubated at 4°C with PL2 (anti-PSGL-1), rapidly washed to remove unbound antibody, and incubated in the absence [PL2-F(ab)2] or in the presence [PL2+F(ab)2] of 10 μg/mL of rabbit anti-mouse IgG F(ab)2 fragments in standard conditions. The anti-CD18 antibody (IB4) was preincubated with PMN together with PL2 for 15 minutes at 4°C before cross-linking. PMN were treated with genistein (10 μg/mL; GEN) for 1 minute after preincubation with PL2 and before cross-linking. In control experiments, PMN were preincubated at 4°C with HC1.1 (anti-CD11c), washed to remove unbound antibody, and incubated in the presence of 10 μg/mL of anti-mouse F(ab)2 fragments in standard conditions [HC1.1+F(ab)2]. PMN aggregation was evaluated as in Fig6. Values are means ± SEM (n = 3). (B) PMN were treated and incubated exactly as for aggregation experiments. The figure shows the Western blot of a representative of three different experiments and the corresponding optical density of P∼110 (means ± SEM, n = 2 to 3).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/93/3/10.1182_blood.v93.3.876/5/blod40325007w.jpeg?Expires=1724223386&Signature=InrHF16LpB2Hc~tgxdQkThgw69HDCwVFREjlvB9m015ekuXxrQXW8gPT9YWtiwqXPjAnWxiuotcusop9oT0x8nLFfDuVk9Ns8x0G8WFSwNtb~w7yOjB77t~EpkK4LJXWTeoOWE9ck8R6qYZMv9~034jDGSLVjAPZwrPSuJQtnNkZY9RfyeTXe0abMrA-Qh1CYMj-pKOcHpTsmhJienYp0mF~U12m0YE8ua8CSIweFT~drpQrEqgPX~6AZifC9-CdTifGapJ-Rf5O~4z52Lo8BNfWnYIAv0b8vtxzd6sX88U65qn-e8nBINOfzL1BKZXBfD1hYU0CvmYuOjfTNDCcLg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Engagement of PSGL-1 by the monoclonal antibody PL2 triggers genistein-sensitive, β2-integrin–dependent PMN aggregation and stimulates protein-tyrosine phosphorylation. (A) PMN were preincubated at 4°C with PL2 (anti-PSGL-1), rapidly washed to remove unbound antibody, and incubated in the absence [PL2-F(ab)2] or in the presence [PL2+F(ab)2] of 10 μg/mL of rabbit anti-mouse IgG F(ab)2 fragments in standard conditions. The anti-CD18 antibody (IB4) was preincubated with PMN together with PL2 for 15 minutes at 4°C before cross-linking. PMN were treated with genistein (10 μg/mL; GEN) for 1 minute after preincubation with PL2 and before cross-linking. In control experiments, PMN were preincubated at 4°C with HC1.1 (anti-CD11c), washed to remove unbound antibody, and incubated in the presence of 10 μg/mL of anti-mouse F(ab)2 fragments in standard conditions [HC1.1+F(ab)2]. PMN aggregation was evaluated as in Fig6. Values are means ± SEM (n = 3). (B) PMN were treated and incubated exactly as for aggregation experiments. The figure shows the Western blot of a representative of three different experiments and the corresponding optical density of P∼110 (means ± SEM, n = 2 to 3).
