Figure 6
Figure 6. SYK is required for normal chemokine signaling and adhesion in vitro and for B-cell migration in vivo. (A) SYK is activated by CXCL12 stimulation of B cells. B cells were stimulated with CXCL12 for the times indicated, and total cell extracts were analyzed by Western blotting with anti-SYK (pY348P). Equal loading of the different samples was confirmed by reprobing the membranes with an antibody recognizing total SYK. The line graph represents densitometry data from 4 experiments showing the ratio of SYK-pY348 to total SYK. The time point at which the SYK-pY348P ratio is significantly elevated above the ratio seen at the 0 time point is marked. (B) SWAP-70 is phosphorylated after CXCL12 stimulation of B cells in a SYK-dependent manner. SWAP-70 underwent IP from B-cell extracts of cells stimulated with CXCL12, and then tyrosine phosphorylation was analyzed by Western blotting. The dependence of this upon SYK activity was ascertained by treating cells with 100nM of the SYK inhibitor Bay61-3606, which inhibited SWAP-70 phosphorylation. Reprobing the blot with anti–SWAP-70 checked equal loading of SWAP-70. (C) SYK is needed for maximal chemotaxis of B cells toward CXCL12. Transwell migration of B cells toward CXCL12 was measured in the presence of increasing doses of the SYK inhibitor Bay61-3606. The percentage of migrated cells was then analyzed. The chart shows mean data from 3 independent experiments performed in triplicate ± SEM (D) Haptotactic migration of B cells through a VCAM-1–coated membrane requires SYK activity. Migration toward CXCL12 was measured in the presence of Bay61-3606. To determine whether there might be any additive effects between SYK inhibition and SWAP70 deficiency both Wt and SWAP70−/− cells were analyzed. The chart shows mean data from 3 independent experiments performed in triplicate ± SEM (E) SYK is required for normal B-cell polarization on anti-CD44. B cells were allowed to attach to anti-CD44–coated surfaces in the presence or absence of Bay61-3606. Images of cells were then taken under polarized light before the F-actin cytoskeleton was stained by the use of rhodamine-phallodin. (F) Less polarized cells are found in the presence of Bay61-3606. The number of cells with a particular morphology was counted (as before in Figure 5). The percentage of polarized, irregular, and round cells with and without SYK inhibition is shown. The chart shows mean data from 3 independent experiments performed in triplicate ± SEM and significance analyzed by t test. A minimum of 100 cells per replicate was counted. (G) SYK inhibition leads to the development of many long dendritic protrusions from the surface of cells. The number of protrusions in each cell counted in panel F was determined. Then, the percentage of cells with 1, 2, 3, or more than 3 protrusions was calculated and plotted, and significance was analyzed by t test. (H) SYK is needed for normal B-cell homing. Purified B cells were labeled with either Cell Tracker Red or Green, and one set of cells was then treated with 100nM Bay61-3606 and the other mock treated and then injected via the tail vein into mice. After 30 or 60 minutes lymph nodes, spleen, and blood were collected and the number and hence ratio of migrated cells (untreated/treated) present was calculated and plotted. 1 represents the input ratio, and the arrows indicate the direction of relative increase of untreated or treated cell numbers. The data shown are from 6 independent experiments with the colors of treated and untreated cells being reversed 3 times. The significance of the difference from the input ratio of untreated/treated cells (1) is shown. (I) T-cell migration is not affected by treatment with Bay61-3606; this panel is the same as panel H except rather than B cells, the migration of T cells was analyzed.

SYK is required for normal chemokine signaling and adhesion in vitro and for B-cell migration in vivo. (A) SYK is activated by CXCL12 stimulation of B cells. B cells were stimulated with CXCL12 for the times indicated, and total cell extracts were analyzed by Western blotting with anti-SYK (pY348P). Equal loading of the different samples was confirmed by reprobing the membranes with an antibody recognizing total SYK. The line graph represents densitometry data from 4 experiments showing the ratio of SYK-pY348 to total SYK. The time point at which the SYK-pY348P ratio is significantly elevated above the ratio seen at the 0 time point is marked. (B) SWAP-70 is phosphorylated after CXCL12 stimulation of B cells in a SYK-dependent manner. SWAP-70 underwent IP from B-cell extracts of cells stimulated with CXCL12, and then tyrosine phosphorylation was analyzed by Western blotting. The dependence of this upon SYK activity was ascertained by treating cells with 100nM of the SYK inhibitor Bay61-3606, which inhibited SWAP-70 phosphorylation. Reprobing the blot with anti–SWAP-70 checked equal loading of SWAP-70. (C) SYK is needed for maximal chemotaxis of B cells toward CXCL12. Transwell migration of B cells toward CXCL12 was measured in the presence of increasing doses of the SYK inhibitor Bay61-3606. The percentage of migrated cells was then analyzed. The chart shows mean data from 3 independent experiments performed in triplicate ± SEM (D) Haptotactic migration of B cells through a VCAM-1–coated membrane requires SYK activity. Migration toward CXCL12 was measured in the presence of Bay61-3606. To determine whether there might be any additive effects between SYK inhibition and SWAP70 deficiency both Wt and SWAP70−/− cells were analyzed. The chart shows mean data from 3 independent experiments performed in triplicate ± SEM (E) SYK is required for normal B-cell polarization on anti-CD44. B cells were allowed to attach to anti-CD44–coated surfaces in the presence or absence of Bay61-3606. Images of cells were then taken under polarized light before the F-actin cytoskeleton was stained by the use of rhodamine-phallodin. (F) Less polarized cells are found in the presence of Bay61-3606. The number of cells with a particular morphology was counted (as before in Figure 5). The percentage of polarized, irregular, and round cells with and without SYK inhibition is shown. The chart shows mean data from 3 independent experiments performed in triplicate ± SEM and significance analyzed by t test. A minimum of 100 cells per replicate was counted. (G) SYK inhibition leads to the development of many long dendritic protrusions from the surface of cells. The number of protrusions in each cell counted in panel F was determined. Then, the percentage of cells with 1, 2, 3, or more than 3 protrusions was calculated and plotted, and significance was analyzed by t test. (H) SYK is needed for normal B-cell homing. Purified B cells were labeled with either Cell Tracker Red or Green, and one set of cells was then treated with 100nM Bay61-3606 and the other mock treated and then injected via the tail vein into mice. After 30 or 60 minutes lymph nodes, spleen, and blood were collected and the number and hence ratio of migrated cells (untreated/treated) present was calculated and plotted. 1 represents the input ratio, and the arrows indicate the direction of relative increase of untreated or treated cell numbers. The data shown are from 6 independent experiments with the colors of treated and untreated cells being reversed 3 times. The significance of the difference from the input ratio of untreated/treated cells (1) is shown. (I) T-cell migration is not affected by treatment with Bay61-3606; this panel is the same as panel H except rather than B cells, the migration of T cells was analyzed.

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