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. 2020 Jan 10;204(4):1012–1021. doi: 10.4049/jimmunol.1901080

FIGURE 4.

FIGURE 4.

ARF6 is copolarized with RPH3A, RAB21, and PIP5K1C90 in ICAM1-stimulated neutrophils. (A–C) Copolarization of active ARF6 with RPH3A (A), RAB21 (B), and PIP5K1C90 (C) in ICAM1 stimulated mouse neutrophils. Neutrophils attached to ICAM1 (1 μg/ml)–coated surfaces were stained with anti-active ARF6 and anti-RPH3A (A), anti-RAB21 (B), or anti-PIP5K1C90 (C), followed with Alexa 488 (green)– and Alexa 633 (red)–conjugated secondary Abs, respectively. Representative optical section images of two cells are shown. The quantification of colocalization is presented as Pearson correlation coefficient (Pearson Coefficient). Each data point represents a cell. The experiments were repeated four times. Scale bar, 3 μm. BF, bright field. (D–F) SecinH3 impairs RPH3A, RAB21, and PIP5K1C90 polarization in mouse neutrophils on the ICAM1-coated surface. Mouse neutrophils were pretreated with DMSO or 20 μM SecinH3 for 20 min at RT and then placed on an ICAM1-coated coverslip for 30 min along with the DMSO or SecinH3 treatment and subjected to immunostaining with anti-RPH3A and anti-TGN38 (D), anti-RAB21 and anti-EEA1 (E), or anti-PIP5K1C90 and anti-EEA1 (F), followed with Alexa 633 (red)– and Alexa 488 (green)–conjugated secondary Abs, respectively. Representative optical section images of two cells are shown. Quantification of polarization of RPH3A, RAB21, and PIP5K1C90 is shown in (D)–(F), respectively. Each data point represents the average of more than 15 cells per observation field, and the experiment was repeated three times. Data are present in means ± SEM (Student t test). ****p < 0.0001.