Binding of GST alone to the different ligands served as the negative control in all experiments. Open in a separate window Figure 3. 2 integrin I domain mediates adhesion to C1q. washing 3 times. Fresh mouse serum at the indicated dilution in PBS was then added to the wells and incubated at 37C for 1 hour. Serum from wild-type or C1q-/- mice, human serum (Sigma-Aldrich), or human serum depleted of the C2 complement component (Sigma-Aldrich) was used for this step. All wells were blocked with BSA (0.1% in PBS) for 1 hour before the addition of cells. PMCs isolated from resident peritoneal exudates using Percoll gradient centrifugation to approximately 85% purity, 6 NMuMg-1 cells, NMuMg-3 cells, K562 cells, or K562 transfectants expressing the human 21 integrin15 were allowed to adhere for 1 hour at 37C in the presence of 2 mM MgCl2 40 nmol PDB or Isosilybin A of 2 mM EDTA. Nonadherent cells were removed by washing, and the remaining adherent cells were quantitated, as previously described.16 SP-A was prepared by the butanol extraction method, as described previously.18 Briefly, 1 to Isosilybin A 2 2 mL alveolar proteinosis was extracted with 25 mL 1-butanol and dried over nitrogen overnight. The dried protein was suspended in HEPES buffer with 0.15 M NaCl and 20 mM n-octyl–antibody, and serum. The suspension was formed by incubation of (1 107 organisms) with anti-antibody (1:200 dilution in 0.3 mL PBS) overnight at 4C, followed by washing and incubation with 50% serum for 1 hour at 37C. The PMC-suspension was centrifuged for 15 minutes and then incubated for 1 hour at 37C. Supernatants were analyzed by ELISA for IL-6 (BD Biosciences). Cloning and manifestation of the 2 integrin I website Cloning and manifestation of the human being 2 integrin subunit I website has been explained elsewhere.19 Briefly, cDNA encoding the 2 2 I domain was amplified Rabbit Polyclonal to OR2G3 by PCR using the full-length 2 integrin cDNA (a gift of Dr Martin E. Hemler). The product of the PCR reaction encodes Ser124-Met349 of the published 2 integrin sequence.20 PCR primers were designed such that a website), or heat-treated undiluted porcine serum (for C1q in Number 3) (Invitrogen Life Systems). Blocking of C1q with BSA only led to an increased background, as evidenced by improved binding of GST-alone (Number S1), likely because the secondary antibodies utilized for detection in the ELISA bound directly to C1q. Therefore, heat-treated porcine serum was used as a obstructing agent for C1q. The porcine serum was warmth treated to degrade the match proteins. Heat-treated porcine serum did not influence 2 integrin I website binding to BSA or type 1 collagen (data not demonstrated). Binding of GST only to the different ligands served as the bad control in all experiments. Open in a separate window Number 3. 2 integrin I website mediates adhesion to C1q. (A-B) The binding of the integrin I website and GST to type 1 collagen (A) or C1q (B) was measured inside a solid-phase binding assay. (C) The binding of the 21 integrin I website, D151A 21 integrin I website mutant, D254A 21 integrin I website mutant, and GST to C1q was measured inside a solid-phase binding assay. (D) The binding of the 2 2 integrin I website, E318A 2 integrin I website mutant, and GST to C1q was measured inside a solid-phase binding assay. All experiments were carried out in the Isosilybin A presence of 2 mM MgCl2. All results are offered as mean SEM from triplicate wells of a single experiment and represent 1 of at least 3 experiments.
