For mRNA isolation, 2.5 × 106 of E15
chick retinal cells were cultured in a
poly-l-ornithine–coated 10-cm culture dish in 13 ml of
Neurobasal with B27 supplement. After 24 hours, the cells were washed
with Neurobasal and further cultured in Neurobasal with 100 ng/ml of
GST–LEDGF or GST, without B27, for 2 days at 37°C. Messenger RNA was
isolated from the cells with a MicroFastTrack 2.0 Kit (Invitrogen,
Carlsbad, CA). The RNA concentration in diethylpyrocarbonate (DEPC;
Sigma)-treated water at 100 ng/ml was measured
spectrophotometrically at 260 nm. Reverse transcription (RT) was
performed in 20 μl of 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 1.5
mM MgCl2, 1.0% Triton X-100, 1 mM of each dNTP,
100 pmol of each specific 3′ primer, 0.5 U/l of RNase inhibitor, 0.25
U/l reverse transcriptase and 100 ng of each mRNA, at 42°C for 1 hour
and then 95°C for 5 minutes. After RT, the reaction mixture was
supplemented with100 pmol of each specific 5′ primer and 5 units of Taq DNA polymerase (Promega, Madison WI) in a volume
of 100 μl of the same buffer as for RT for use in PCR. After
denaturation for 5 minutes at 94°C, 15, 20, 25, and 30 cycles of PCR
amplification (denaturation at 94°C for 30 seconds, annealing at
55°C for 20 seconds, elongation at 72°C for 20 seconds) were
carried out, followed by final extension for 5 minutes at 72°C. The
PCR products were electrophoresed in a 1.3% agarose gel and visualized
by ethidium bromide staining. The value of each band was measured with
image-analysis (C-80 Epi-Illumination UV Darkroom, New England
Scientific Associates, Salem, NH, and Scion Image 1.62, NIH). The
primer sets were purchased from GIBCO BRL. The sequences of
oligonucleotide probes were as follows: Hsp90, 5′ primer,
5′-ACTTTTGTCTGCATTCCCTC, bp 2310 to 2329, and 3′ primer,
5′-GAACACCCAGATGTCATACC, bp 2543 to 2562.