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. Author manuscript; available in PMC: 2017 Aug 8.
Published in final edited form as: Cell Rep. 2017 Jun 20;19(12):2586–2597. doi: 10.1016/j.celrep.2017.05.080

Figure 3. RAB21 is polarized and involved in PIP5K1C90 polarization in neutrophils.

Figure 3

A–C. RAB21DN mutant inhibits PIP5K1C90 polarization in mouse neutrophils. Mouse neutrophils were cotransfected with HA-PIP5K1C90 and GFP (A) or RAB21DN-GFP (B) and sorted by FACS. GFP-positive cells were stimulated by Fb for 30 min and stained by anti-HA and anti-EEA1 antibodies, followed by secondary antibodies conjugated with Alexa633 (colored in red) and Alexa488 (colored in green). Two representative cells are shown in A and B. C shows the quantification of the percentage of neutrophils showing PIP5K1C90 polarization from three independent experiments. More than 20 cells were examined in each experiment. Data are presented as means ± SD (*, p<0.01, Student’s t-Test). Fig S2A shows localization of PIP5K1C90 and EEA1 staining in representative cells placed on PK-coated surfaces as a control.

D–F. Polarization of RAB21 in mouse neutrophils upon Fb stimulation. Mouse neutrophils were placed on coverslips coated with PK (D) or Fb (E) for 30 min and stained by anti-RAB21 and Alexa-488-conjugated secondary antibody (green) in presence of Alexa647-phalloidin (red). F shows the quantification of the percentage of neutrophils showing RAB21 polarization from three independent experiments. More than 20 cells were examined in each experiment. Data are presented as means ± SD (*, p<0.01, Student’s t-Test).

G. Polarization of RAB21 and PIP5K1C90 in neutrophils. Mouse neutrophils transfected with HA-PIP5K1C90 were placed on Fb-coated coverslips for 30 min and stained by anti-HA and anti-RAB21 antibody followed by secondary antibodies conjugated with Alexa633 (colored in red) and Alexa488 (colored in green). Fig S2C shows localization of PIP5K1C90 and RAB21 staining in cells placed on PK-coated surfaces as a control.

H. PKN1-deficiency impairs RAB21 polarization. Mouse neutrophils were placed on Fb-coated coverslips for 30 min and fixed and stained by anti-RAB21 and anti-EEA1 antibodies, followed by secondary antibodies conjugated with Alexa633 (colored in red) and Alexa488 (colored in green). The quantification for percentage of neutrophils showing RAB21 polarization was done from three independent observations, and more than 20 cells were examined in each observation. Data are presented as means ± SD (*, p<0.01, Student’s t-Test).