Ghafar et al
Buttyan R, Jacobs BZ, Blaivas J, and Levin RM (1992). The
early molecular response to rabbit bladder outlet obstruction.
Neurourol Urodyn 11:225–238.
Protein Extraction and Western Blotting for HIF-1␣,
VEGF-A, Angiopoietin-1, or Endostatin
Frozen tissues were pulverized under liquid nitrogen
and the frozen powder was then thawed and homog-
enized (using a Polytron tissue homogenizer; Brink-
mann Instruments, Inc., Westbury, New York) in 5
volumes of SDS buffer (120 mM Tris-HCL, pH 6.8, 4%
SDS). The homogenate was stored on ice for 40
minutes before the removal of nuclear and other
debris by centrifugation at 14000 ϫg for 15 minutes.
Protein concentrations in the extracts were deter-
mined using the Bio-Rad Protein Assay system (Bio-
Rad Labs, Inc., Richmond, California). Aliquots of
extracts containing 50 gs of protein were solubilized
in loading buffer (10% glycerol, 2% SDS, 60 mM Tris,
pH 6.8, 0.01% bromophenol blue, and 100 mM DTT)
and were boiled for 5 minutes before electrophoresis
on 12% SDS-polyacrylamide gels. The proteins in the
gel were electrophoretically transferred to nitrocellu-
lose membranes (Hybond; Amersham, Inc., Arlington
Heights, Illinois) to produce Western blots in 25 mM
Tris-HCL, pH 8.0, 192 mM Glycine, and 20% methanol
at 100 volt for 1 hour. Membranes were blocked in
10% nonfat milk powder (in TBS-T, 20 mM Tris-HCL,
pH 7.6, 136 mM NaCl, 0.3% Tween-20) at 4° C over-
night and were then incubated for 1 hour with a mouse
monoclonal antibody against human HIF-1␣ (diluted
1:1000; Novus Biologicals, Littleton, Colorado), or with
mouse monoclonal antibody against rat VEGF (Santa
Cruz Biotechnology, Inc., Santa Cruz, California) di-
luted 1:100 in TBS-T buffer at 37° C (for detection of
VEGF), or with goat polyclonal antibodies against rat
Angiopoietin-1 (diluted 1:200, Santa Cruz Biotechnol-
ogy, Inc.) or goat polyclonal antibody against rat en-
dostatin (diluted 1:500; a gift from Dr. Jonathan Barach).
After extensive washing with TBS-T buffer, membranes
were incubated with a secondary antibody (sheep anti-
mouse IgG-horseradish peroxidase complex for VEGF
and HIF-1␣ or goat anti-goat IgG-horseradish peroxi-
dase for angiopoietin-1 or endostatin; Santa Cruz Bio-
technology, Inc.). Chemoluminescent detection of anti-
body binding was performed using the ECL Western
blotting analysis system (Amersham, Inc.). Subsequently
all Western blots were cleaned by incubation in a dena-
turing buffer (0.2% SDS, 0.1 mM DTT) at 70 ° for 30
minutes followed by several washings in TBS-T and
reblocking with 10% milk solution. The blot was then
reprobed for smooth muscle actin using (mouse mono-
clonal diluted 1:2000; Sigma Chemical Company, Inc.,
St Louis, Missouri). The autoradiograph was analyzed
with the Molecular Dynamics Scanning Densitometer
(Sunnyvale, California).
Chen MW, Buttyan R, and Levin RM (1996). Genetic and
cellular response to unilateral ischemia of the rabbit urinary
bladder. J Urol 155: 732–737.
Chichester P, Lieb J, Levin SS, Buttyan R, Horan P, and Levin
RM (2000). Vascular response of the rabbit bladder to short
term partial outlet obstruction. Mol Cell Biochem 208:19–26.
Chichester P, Schroder A, Horan P, and Levin RM (2001).
Vascular response of the rabbit bladder to chronic partial
outlet obstruction. Mol Cell Biochem 226:1–8.
Kato K, Wein AJ, Kitada S, Haugaard N, and Levin RM (1988).
The functional effect of mild outlet obstruction on the rabbit
urinary bladder. J Urol 140:880–884.
Kato K, Wein AJ, Longhurst PA, Haugaard N, and Levin RM
(1990). Effects of outlet obstruction on glucose metabolism
of the rabbit urinary bladder. J Urol 143:600–606.
Kim JC, Yoon JY, Seo SI, Hwang TK, and Park YH (2000).
Effects of partial bladder outlet obstruction and its relief on
types I and III collagen and detrusor contractility in the rat.
Neurourol Urodyn 19:29–42.
Levin RM, Brading AF, Mills IW, and Longhust PA (1999).
Prostatic disease. St. Louis: WB Saunders Co.
Levin RM, Haugaard N, O’Connor L, Buttyan R, Das A, Dixon
JS, and Gosling JA (2000). Obstructive response of human
bladder to BPH vs. rabbit bladder response to partial outlet
obstruction: A direct comparison. Neurourol Urodyn 19:609–
629.
Levin RM, Longhurst PA, Barasha B, McGuire EJ, Elbadawi
A, and Wein AJ (1992). Studies on experimental bladder
outlet obstruction in the cat: Long-term functional effects.
J Urol 148:939–943.
Levin RM, Longhurst PA, Kato K, McGuire EJ, Elbadawi A,
and Wein AJ (1990a). Comparative physiology and pharma-
cology of the cat and rabbit urinary bladder. J Urol 143:848–
852.
Levin RM, Longhurst PA, Monson FC, Kato K, and Wein AJ
(1990b). Effect of bladder outlet obstruction on the morphol-
ogy, physiology, and pharmacology of the bladder. Prostate
3(Suppl):9–26.
Levin RM, Memberg W, Ruggieri MR, and Wein AJ (1986).
Functional effects of in vitro obstruction on the rabbit urinary
bladder. J Urol 135:847–851.
Lieb JI, Chichester P, Kogan B, Das AK, Leggett RE, Schro-
der A, and Levin RM (2000). Rabbit urinary bladder blood
flow changes during the initial stage of partial outlet obstruc-
tion. J Urol 164:1390–1397.
Radzinski C, McGuire EJ, Smith D, Wein AJ, Levin RM, Miller
LF, and Elbadawi A (1991). Creation of a feline model of
obstructive uropathy. J Urol 145:859–863.
References
Arteel GE, Thurman RG, and Raleigh JA (1998). Reductive
metabolism of the hypoxia marker pimonidazole is regulated
by oxygen tension independent of the pyridine nucleotide
redox state. Eur J Biochem 253:743–750.
Rohner RJ Jr, Hannigan JD, and Sanford EJ (1978). Altered in
vitro adrenergic responses of dog detrusor muscle after
chronic bladder outlet obstruction. Urology 11:357–361.
Saito M, Wein AJ, and Levin RM (1993). Effect of partial outlet
obstruction on contractility: Comparison between severe and
mild obstruction. Neurourol Urodyn 12:573–583.
Blaivas JG (1996). Obstructive uropathy in the male. Urol Clin
North Am 23:373–384.
908 Laboratory Investigation • July 2002 • Volume 82 • Number 7