![Neutrophil DREAM enhances the ligand-binding function of β2 integrin through activated IKKβ.(A–C) WT and DREAM KO neutrophils were treated with or without 5 ng/ml TNF-α or 10 µM fMLP for 10 min unless otherwise stated. Flow cytometry was performed with anti-αMβ2 or anti-αLβ2 antibodies, and DyLight 488–conjugated Fg. (D) WT and DREAM KO neutrophils were placed on immobilized ICAM-1 in the presence or absence of TNF-α or fMLP. Adherent cells were counted under a microscope. (E–J) WT and DREAM KO neutrophils were pretreated with or without 0.1% DMSO (− or vehicle [Veh]), TPCA-1 (an IKKβ-selective inhibitor, 0.1–1 µM), or Bay 11–7082 (Bay; an IKKα/β inhibitor, 1–3 µM) and then incubated with or without TNF-α or fMLP for (E–G) 5 or (H–J) 10 min. (E–G) Lysates were immunoblotted with antibodies against pIKKα/β or total IKKβ and subjected to densitometric analysis (mean ± SD, n = 3). (H–J) Flow cytometry was performed with anti-αMβ2 or anti-αLβ2 antibodies. (I) TPCA-1: 0.3 µM and Bay: 3 µM. (K–N) dHL-60 cells were treated with or without TNF-α for 10 min and subjected to flow cytometry with antibodies against activated αMβ2 (CBRM1/5), total αMβ2 (ICRF44), extended αLβ2 (NKI-L16), or activated β2 (mAb24). (O–R) Human neutrophils were pretreated with 0.1% DMSO (Veh) or 0.3 µM TPCA-1, stimulated with TNF-α, and used for flow cytometry. The MFI values were measured. (S and T) WT neutrophils were treated with or without fMLP or TNF-α for 0–5 min. Lysates were immunoprecipitated with control IgG or anti-β2 antibodies and subjected to immunoblotting with antibodies against talin1 or β2 and densitometric analysis. (U and V) WT or DREAM KO neutrophils were pretreated with or without vehicle or TPCA-1 and stimulated with fMLP or TNF-α. Talin1-β2 binding was detected as described above. Data represent the mean ± SD (n = 3 or 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 after ANOVA and either Dunnett’s test (E–G, versus vehicle control [+ TNF-α], and S and T, versus unstimulated control [−]) or Tukey’s test. IP, immunoprecipitation; Scr, scrambled.](https://cdn.rupress.org/rup/content_public/journal/jem/219/1/10.1084_jem.20211083/4/jem_20211083_fig4.png?Expires=1771551902&Signature=frXHN4VtuWMJ8y8nyXV87p7AiDIa2jeCzkUHRpqRO1UnqxF3-MJUu4u3s7JxTIsICvSUSJf2TiomAaEuXBhDfa~enqGKVHOca0zdFnsvweSCRmjwFKcXHcMR3lwJEFZQPzGu4F66tVI5uXiNDKTWKsdg3WfyrhheQvt9prCv0A~vBdt0RjNjvkqcFZ~VGcEBTmN6AR6XGJ-2wqo54NIkOi2-yomyqMonzCFUkGh-ktk0nol2zcC2rh6LlPfkStKxtZFi89PPRCopi759ZXeg9Wkcn5W1sdSQCRRJTx52ZjcaQjHcgh5QQpuxS3POuk~heQ8Z4EwlK0f9-xi2D4fUfQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Neutrophil DREAM enhances the ligand-binding function of β2 integrin through activated IKKβ. (A–C) WT and DREAM KO neutrophils were treated with or without 5 ng/ml TNF-α or 10 µM fMLP for 10 min unless otherwise stated. Flow cytometry was performed with anti-αMβ2 or anti-αLβ2 antibodies, and DyLight 488–conjugated Fg. (D) WT and DREAM KO neutrophils were placed on immobilized ICAM-1 in the presence or absence of TNF-α or fMLP. Adherent cells were counted under a microscope. (E–J) WT and DREAM KO neutrophils were pretreated with or without 0.1% DMSO (− or vehicle [Veh]), TPCA-1 (an IKKβ-selective inhibitor, 0.1–1 µM), or Bay 11–7082 (Bay; an IKKα/β inhibitor, 1–3 µM) and then incubated with or without TNF-α or fMLP for (E–G) 5 or (H–J) 10 min. (E–G) Lysates were immunoblotted with antibodies against pIKKα/β or total IKKβ and subjected to densitometric analysis (mean ± SD, n = 3). (H–J) Flow cytometry was performed with anti-αMβ2 or anti-αLβ2 antibodies. (I) TPCA-1: 0.3 µM and Bay: 3 µM. (K–N) dHL-60 cells were treated with or without TNF-α for 10 min and subjected to flow cytometry with antibodies against activated αMβ2 (CBRM1/5), total αMβ2 (ICRF44), extended αLβ2 (NKI-L16), or activated β2 (mAb24). (O–R) Human neutrophils were pretreated with 0.1% DMSO (Veh) or 0.3 µM TPCA-1, stimulated with TNF-α, and used for flow cytometry. The MFI values were measured. (S and T) WT neutrophils were treated with or without fMLP or TNF-α for 0–5 min. Lysates were immunoprecipitated with control IgG or anti-β2 antibodies and subjected to immunoblotting with antibodies against talin1 or β2 and densitometric analysis. (U and V) WT or DREAM KO neutrophils were pretreated with or without vehicle or TPCA-1 and stimulated with fMLP or TNF-α. Talin1-β2 binding was detected as described above. Data represent the mean ± SD (n = 3 or 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 after ANOVA and either Dunnett’s test (E–G, versus vehicle control [+ TNF-α], and S and T, versus unstimulated control [−]) or Tukey’s test. IP, immunoprecipitation; Scr, scrambled.
