Plates (100-mm) were washed once with cold binding buffer (DMEM
with 0.1% bovine serum albumin [BSA] and 25 mM HEPES) and then
incubated at 37°C for two washes, 1 hour each, allowing dissociation
of bound endogenous TGF-β from the cells and from the tissue culture
plastic.
23 24 Cells were incubated with 80 to 400 pM
125I-TGF-β1 (NEN Life Sciences, Boston, MA). Of
five independent experiments performed, incubation with 150 pM
125I-TGF-β1 was repeated three times. The
dishes were placed in the 5% CO
2 incubator for 5
minutes and then sealed with parafilm and incubated with shaking for 2
to 3 hours at 0°C to 4°C. To terminate the incubation, we collected
the medium and washed the cells twice with Hanks’ balanced salt
solution (HBSS) buffered with 40 mM HEPES, pH 7.4 (Sigma), followed by
incubation for 30 minutes at 4°C with the covalent cross-linking
reagent, disuccinimidyl suberate (Pierce, Rockford, IL) in dimethyl
sulfoxide at a final concentration of 0.3 mM in HBSS. The cross-linking
reaction was quenched by washing three times with cold 250 mM sucrose,
10 mM Tris (pH 7.4), and 1 mM EDTA. The cells were lysed in 4× sodium
dodecyl sulfate (SDS) sample buffer at a dilution to yield 1× SDS
sample buffer. Protein was assayed by a highly sensitive quantitative
colorimetric determination (Micro BCA, Pierce), and 20 μg per
lane was electrophoresed in 10% SDS-polyacrylamide gel
elecctrophoresis (PAGE) under reducing conditions along with prestained
molecular size markers. After drying, the gel was exposed to film
(Biomax MS; Kodak) with an intensifying screen for 1 to 3 days and
developed. The relative amount of
125I-TGF-β1 bound to
receptors I and II (RI and RII) was determined by scanning the
autoradiogram with a flatbed scanner and analyzing signal over
background using the image analysis program.
To determine the specificity of TGF-β1 binding, cells were incubated
as above with 150 pM
125I-TGF-β1 plus a 10-fold excess of
unlabeled TGF-β1, 1.5 nM.
23