FIGURE 1.

RPH3A and RAB21 polarizations are compromised in siARF6-transfected mouse neutrophils. (A–D) ARF6-knockdown impairs RPH3A and RAB21 polarization. Mouse neutrophils were cotransfected with GFP plasmid and siCtr or siARF6 and cultured for 48 h; GFP-positive cells were sorted out by FACS and attached onto Fb-coated surface before being stained by anti-RPH3A and anti-TGN38 (A) or anti-RAB21 and anti-EEA1 (B), followed with Alexa 633 (red)– and Alexa 488 (green)–conjugated secondary Abs, respectively. Representative optical section images of two cells are shown (A and B). Quantification of polarization was performed as described in the Materials and Methods and is shown in (C) and (D). Each data point represents the average of more than 10 cells per observation field, and the experiment was repeated four times. Data are present in means ± SEM (Student t test). TGN38 is a resident integral membrane protein of the TGN and thus used as a marker for TGN. Scale bar, 3 μm. ****p < 0.0001.