Fig 2: CD40 interacts with CD11a. Sorted cells were Mn2+ activated, or not, then immunoprecipitation was performed with anti-CD40 antibody 4F11. A, Western blot for CD11a was performed. B, fold change of band intensities in response to Mn2+ was analyzed, and significant differences were calculated by one-tailed t test (for CD11a) or two-tailed t test (for CD40). Significant p-values are shown in the figure. Data are representative of three different experiments.

Fig 3: CD4 negative and Th40 cells alter CD40 and CD18 surface expression in response to Mn2+. PBMCs were stained for CD4, CD40, CD18, CD11a, and CD11b and analyzed by flow cytometry. In the lymphocyte gate, cells were gated on CD4-negative (CD4neg), CD4lo, or CD4hi, then the expression of CD40 (A) and CD18 (B) was analyzed. Tinted histogram, isotype; solid line, no activation; dotted line, Mn2+ activation. p-Values were calculated, in A, by two-tailed t test and, in B, by one-way ANOVA with Tukey's multiple comparisons test.

Fig 4: 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 5: 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.