Fig 2: KGYY15interacts with CD11a, CD11b, and CD18. Sorted NOD and BALB/c cells were activated with Mn2+, or not, then immunoprecipitation was performed with KGYY15-conjugated microbeads. A, Western blots for CD11a (top panels), CD11b (second from top panels), CD18 (third from top panels), and CD40 (bottom panels) were performed. B and C, fold change of band intensities in response to Mn2+ was analyzed, and significant differences were calculated by one-tailed t test (for CD11a, CD11b, and CD18; except for BALB/c PBMC large CD11a band where two-tailed test was used) or two-tailed t test (for CD40). Significant p-values are shown in the figure. Data are representative of least three different experiments.

Fig 3: KGYY15interacts with recombinant CD40 and CD11a/CD18 or CD11b/CD18 proteins in KinExAs. KGYY15 interactions were assayed by KinExA solution phase assay with recombinant CD40, CD11a/CD18, CD11b/CD18, or combinations of CD40 and one or the other integrin. A, KGYY15 with CD40; Kd = 109.69 nM. B, KGYY15 with CD11a/CD18+CD40; Kd = 166.78 nM. C, KGYY15 with CD11b/CD18+CD40; Kd = 7.09 nM.

Fig 4: Glycosylation accounts for the different size bands of CD11a, CD11b, and CD18.A, NOD cell lysates were either assayed as is (NOD whole cell lysates) or subjected to immunoprecipitation by an OVA-peptide (OVA-pep IP; a control for KGYY15 peptide) or by CD3 antibody (CD3-ab IP; a control for CD40 antibody 4F11) then assayed for CD11a, CD11b, CD18, or CD40 in Western blots. CD3-ab immunoprecipitation eluates were also assayed for CD3. B, NOD PBMC lysates were subjected to deglycosylation by PNGase F (removal of N-linked deglycosylation) or by PNGase F and total O-linked deglycosylation (Total deglyc.), or not (Lysates), then Western blots for CD11a, CD11b, and CD18 were performed. Completely deglycosylated bands are indicated (compl. deglyc.). C, lysates were subjected to deglycosylation by PNGase F alone, PNGase F and total O-linked deglycosylation (Total deglyc.), deglycosylation with four different enzymes that remove O-linked glycosylations (O-linked deglyc.), or the four different enzymes that remove O-linked glycosylations separately (O-glycosidase, neuraminidase, ß-galactosidase, or glucosaminidase). Western blots were then done for CD11a. Data are representative of three experiments.

Fig 5: KGYY15binding is partially blocked by antibodies to CD11a and CD11b as well as by recombinant CD11a/CD18, CD11b/CD18, and CD40 proteins, but not recombinant CD28 protein. NAMALWA cells were stained with KGYY15-FITC in the absence/presence of competition. A, competition with anti-CD11a, anti-CD11b, or recombinant CD11a/CD18 or CD11b/CD18 proteins. B, competition with recombinant CD40 protein. C, competition with recombinant CD28 protein. Data are represented as mean ± SEM. p-Values were calculated by two-tailed t test and are shown in the figure.