(A) Amino acid sequences of the cytoplasmic domains of wildtype and mutant PSGL-1 proteins used in the coimmunoprecipitation experiments. The previously identified ERM interaction motif is underlined.[61] (B) SLIC-1 and wildtype or mutant PSGL-1 were coexpressed in COS cells. Cell extracts were subjected to immunoprecipitation with a mouse monoclonal anti-PSGL-1 antibody. The presence of SLIC-1 and PSGL-1 in the immunocomplex was analyzed by Western blot analysis following reducing SDS-PAGE (Top 4 panels). The total levels of over-expressed SLIC-1 and PSGL-1 in the cell extracts were analyzed by Western blot analysis (bottom four panels). SLIC-1 runs higher on SDS-PAGE gel (slightly above 43kDA molecular weight marker) than its predicted molecular weight (36kDa). The reason for this molecular weight shift remains unclear. PSGL-1 appears as a ladder of bands due to its extensive post-translational modification.[62] * denotes background IgG bands. (C) A total of 45 12-amino acid peptides representing overlapping sequences in the cytoplasmic domain of PSGL-1 were synthesized onto cellulose membranes, which were probed with indicated GST fusion proteins expressed and purified from E. Coli. Peptides that showed positive interactions are numbered on the right. Deduced amino acid sequences for each interaction motif are shown beneath each panel. (Sections relating to 2C.a and 2D deleted).

(A) Sequences of PX domains from different proteins were aligned using CLUSTAIW program. Most, moderately, and less conserved residues are shown in yellow letters on green background, white letters on green background, and black letters on yellow background, respectively. (B-D) Wildtype and R116Q mutant SLIC-1 PX domain fused C-terminally to GST were expressed and purified from E. Coli. The levels of these proteins were examined on a coomossie-stained SDS-PAGE gel (D). Their lipid binding activities were tested in a lipid overlay assays using commercial lipid strips (B) or lipid arrays (C). Concentration of each lipid spot on the lipid strip was 100pmol. Concentrations of lipids used for spotting each lipid array were 100pmol, 50pmol, 25pmol, 12.5pmol, 6.3pmol, 3.2pmol, and 1.6pmol. LPA, lysophosphatidic acid; LPC, lysophosphocholine; S1P, sphingosine-1-phosphate; PE, phosphatidylethanolamine; PA, phosphatidic acid; PC, phosphatidylcholine; PS, phosphatidylserine, PI, phosphoinositide.

(A) CHO cells stably expressing PSGL-1 were transfected with pEGFP-C1-SLIC-1 (a, b, c) or pEGFP-C1-SLICR116Q (d, e, f). In 24 hr after transfection, cells were fixed, permeabilized, and subjected to indirect immunofluorescent staining with a mouse anti-PSGL-1 monoclonal antibody and Texas Red-conjugated goat anti-mouse antibody. Confocal microscopy was performed to visualize PSGL-1 staining in red (a, d), and EGFP-SLIC-1 (b) or EGFP-SLICR116Q (e) in green. Panels c and f represented merged images. Experiment was repeated four times and similar results were obtained. (B) One hundred cells from one representative experiment in A were counted under fluorescence microscope. Percentage of cells that showed PSGL-1 staining in vesicles among transfected cells (green cells) was calculated and shown in the bar graph. (C) COS cells were transfected with plasmid expressing PSGL-1 in the presence or absence of SLIC-1. Cell lysates were separated by SDS-PAGE and Western analysis was carried out to determine the total levels of PSGL-1.

Murine neutrophils were labeled were perfused over an L-E/I monolayer, allowed to roll and arrest, and then labeled with anti-PSGL-1 conjugated to PE. Distribution of PSGL-1 was imaged by immunofluorescent microscopy. (A, B) Clusters of PSGL-1 (defined as regions 2.5 standard deviations higher than background) covered the same total surface area and had the same distribution of distances from the cell centroid between SNX20 −/− and wildtype neutrophils rolling on an L-E/I monolayer. (C) There was no significant difference in the average number of clusters between the two cell types. (D) Sample images show a representative image of PSGL-1 distribution on the surface of a rolling neutrophil. Data was based on a total of 290 neutrophils for each condition. There was no significant difference between the average values for wildtype and SNX20 −/− neutrophils in cluster size, number of clusters, or cluster distance from centroid.

(A) Nucleotide and predicted amino acid sequences of human SLIC-1. The PX domain is shaded. PXXP motifs are in bold. The amino acid sequences obtained from the yeast two-hybrid screen are underlined. (B) The amino acid sequences of human SLIC-1 and its mouse homologue SNX20 as well as a homologous human protein SNX21/SNX-L were aligned using the CLUSTALW program. Residues completely conserved in all three proteins are shaded in black. Those completely conserved in two proteins are shaded in dark gray. Moderately conserved residues are shaded in light gray.

COS cells were transfected with EGFP-SLIC1. For EEA1 co-staining, cells were fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton-X. Indirect immunofluorescent staining was carried out using a mouse anti-EEA1 monoclonal antibody and Texas Red-conjugated goat anti-mouse antibody. A representative COS cell expressing EGFP-SLIC-1 is shown in green (A). EEA1 staining of the same cell is shown in red (B). Yellow in C indicates co-localization of SLIC-1 and EEA1 when images A and B are merged. For transferrin labeling, cells were starved for 3 hr before they were labeled with Texas Red-conjugated transferrin for 15 min. Cells were fixed and confocal microscopy was performed to analyze co-localization. One representative COS cells expressing EGFPSLIC-1 is shown in green (D). Transferrin labeling of the same cell is shown in red (E). Yellow in F indicates co-localization of SLIC-1 and transferrin when images D and E are merged.

(A) COS cells were transfected with pEGFP-C1-SLIC-1 or pEGFP-C1-SLICR116Q. In 24 hr post transfection, cells were fixed and subjected to confocal microscopy. Top panel: One representative COS cells expressing EGFP-SLIC-1; Bottom panel: Three representative COS cells expressing EGFP-SLICR116Q. (B) COS cells transfected with EGFP-SLIC-1 were treated with Wortmannin for indicated periods of time and analyzed by fluorescent microscopy. Bright staining shows EGFP-SLIC-1 positive cells.

Murine bone marrow neutrophils were perfused over a cell monolayer expressing E-selectin and ICAM-1, and their subsequent rolling and arrest was recorded by phase contrast videomicroscopy. (A, B) There was no significant difference (p>0.05) between the number of wildtype or SNX20 deficient neutrophils that rolled or transitioned to arrest in an average microscope field under 1 dyne/cm^2 shear stress. (C) When exposed to 0.1 nM MIP1a, SNX20 −/− and wildtype neutrophils were equally capable of transitioning from rolling to firm arrest with high efficiency. Data was representative of 6 experimental runs, each of which was the average of 6 microscope fields over the course of 6 minutes.

Murine neutrophils were loaded with calcium sensitive Fluo-4 dye, treated with anti-PSGL-1 and a secondary crosslinker, then analyzed for calcium flux by flow cytometry. The fraction of Fluo-4 stained neutrophils above an intensity threshold was plotted for SNX20 −/− and wildtype neutrophils after PSGL-1 or control antibody crosslinking treatment over a period of 10 minutes. After 160 seconds, both SNX20 −/− and wildtype neutrophils exhibited greater calcium flux than the unstimulated controls. There was no significant difference in the calcium flux between SNX20 −/− or wildtype neutrophils, while the conditions marked with * exhibited significantly more calcium flux (p<0.05) than the respective neutrophil type (WT or SNX −/−) in the absence of antibody crosslinking (control IgG).