Total RNA was isolated by the method of Chomczynski and
Sacchi
32 after tissue homogenization (Polytron). RNA (15μ
g) was electrophoresed through a 1% agarose-formaldehyde gel and
transferred to nylon filters (Gene Screen Plus; New England Nuclear,
Boston, MA). The filters were prehybridized in buffer containing 50%
deionized formamide, 5× SSPE, 5× Denhardt’s solution, 0.5% sodium
dodecyl sulfate (SDS), 10% dextran sulfate, and denatured salmon sperm
DNA (100 μg/ml) and hybridized at 42
oC in fresh
buffer without salmon sperm DNA. The hybridization buffer contained
either a 520-bp
NcoI/
BglII fragment of the human
VEGF cDNA (gift of Herbert Weich), a 226-bp
EcoRI/
BamHI fragment of human VEGFR-1, or
a 286-bp fragment of human VEGFR-2.
33 The blots were
stripped and reprobed with a 400-bp fragment encompassing the 3′
untranslated region of the human β-actin cDNA. The cDNA probes were
labeled with a random primed DNA labeling kit using[α
-
32P] deoxy-CTP (Boehringer Mannheim, Indianapolis,
IN). Filters were washed in 2×–0.5× SSPE, 0.1% SDS, for varying
times and at increasing temperatures. The washes were titrated for
maximum signal-to-noise ratio. The hybridized and washed filters were
exposed to x-ray film (X-Omat AR; Eastman Kodak , Rochester, NY) with
an intensifying screen at −70
oC for 12 to 72 hours.
Densitometry was performed on all blots and normalized to the
corresponding actin signal for each lane using a digital imaging system
(IS-1000 with ver. 1.97 software; Alpha Inotech, Torrence, CA).