After washing with PBST, HRPO-streptavidin (1:5000; Vector Laboratories, Burlingame, CA, USA) or HRPO-conjugated goat anti-mouse IgG (1:5000; Biosource, Camarillo, CA, USA) in 10 mM TBS (pH 7.2) was then added and reacted for 30 min at room temperature. After washing learn more with PBST, the wells were subjected to color development
by the addition of 0.1 ml of 0.91 mM 2,2′azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) in 0.1 M citrate (pH 4.1) containing 0.04% (v/v) H2O2. The reaction was stopped by the addition of 0.1 ml of 0.1 M citric acid containing 0.01% (w/v) NaN3. The absorbance at 405 nm was then measured in a microplate reader (SpectraMax 340 C, Molecular Devices, Sunnyvale, CA, USA). Fn or rFbp (each at 1 mg/ml) were incubated
with 0.1 mM biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sigma) in VBS for 1 hr at room temperature. After incubation, a one-fifth volume of 0.5 M Tris-glycine buffer (pH 7.5) was added and the mixture was then further incubated for 1 hr at room temperature. Unattached biotin was removed using a desalting column (GE Healthcare). A plate binding assay was carried out by coating the wells with Fn fragments (70 kDa, 30 kDa, 45 kDa, 110 kDa or III1-C) and by assay of the binding of biotinylated rFbpA or biotinylated rFbpB in BVBS containing 0.02% (v/v) Tween 20. Both rFbpA-Sepharose and rFbpB-Sepharose were prepared www.selleckchem.com/products/chir-99021-ct99021-hcl.html by coupling NHS-activated Sepharose (GE Healthcare) with rFbpA and rFbpB respectively, according to the instruction manual. Both rFbpA-Sepharose and rFbpB-Sepharose were applied with 25 mg and 30 mg Fn respectively. Bound proteins were then eluted with 4 M urea in VBS. The resulting eluates were designated as rFbpA-BP and rFbpB-BP, respectively. A plate binding assay was carried out by coating the wells with Fn fragments (70
kDa, 30 kDa, 45 kDa, 110 kDa, or III1-C) or with Fn and by assay of binding of the anti-Fn mAbs HB91, HB39, ZET1, or ZET2. Samples containing Forskolin rFbpA-BP, rFbpB-BP or Fn were mixed with an equal volume of Laemmli sample buffer. Proteins were separated on a 7% SDS-PAGE gel under non-reducing conditions. The electrophoresed components were then either subjected to silver staining or transferred from the gel to a PVDF membrane (Millipore, Billerico, MA, USA) using a transblot unit (Atto, Tokyo Japan). The transblotted PVDF membrane was blocked with casein blocking buffer (Sigma) for 2 hr at room temperature and then incubated with 20 ml of anti-Fn mAbs (0.01 mg/ml) in VBS containing 10% casein blocking buffer for 1 hr at room temperature. After washing with PBST, the membrane was reacted HRPO-conjugated goat anti-mouse IgG (1:5000) in TBS for 30 min at room temperature. After washing with PBST, the membrane was subjected to color development with 0.25 mg/ml 3,3′-diaminobenzidine (Sigma) in 50 mM Tris-HCl, pH 8.0, containing 0.01% (v/v) H2O2.