Reverse transcription quantitative PCR
The cDNA was synthesized using Moloney murine leu-
kemia virus (M-MLV) kit (28025013; ThermoFisher,
Waltham, MA, USA). A Real-time system (q-PCR) was
used to detect PPV interrelated mRNA, β-actin as an
internal control. All primer in Table
1
were synthesized
(Sangon Biotech, Shanghai, China).
Immunofluorescence assay and confocal imaging
Immunofluorescence assay was operated as previously
described [
32
]. The cells were incubated with the pri-
mary antibodies 3C9, SYNCRIP or Cy3-NS1 mRNA Fish
probe. After further washing, the cells were incubated
with the secondary antibodies for 1 h at room tempera-
ture in the dark. After the final washing, the cover glasses
were removed from the wells and fixed onto a glass slide,
and the image was captured by a confocal microscopy.
RNA‑immunoprecipitation assay
These assays were done according to the Abcam RNA-
immunoprecipitation manufacturer. Briefly, cells were
harvested by trypsinization and resuspended in PBS,
2 mL freshly prepared nuclear isolation buffer was
added (1.28 M sucrose, 40 mM Tris–HCl pH 7.5, 20 mM
MgCl
2
, 4% Triton X-100) and the sample was kept on ice
for 20 min. Then the cells were centrifuged at 2500 g for
15 min to obtain a pelleted nuclei. The nuclear pellet was
resuspended in freshly prepared RIP buffer (150 mM KCl,
25 mM Tris pH 7.4, 5 mM EDTA, 0.5% NP40), 1 mM
PMSF, 100 U/mL RNase inhibitor, as well as 1 × protease
inhibitor cocktail (Sigma). Centrifugation was then used
to obtain a pellet containing the nuclear membrane and
debris at 13 000 g for 10 min. Then the specific antibody
and control antibody IgG were added for 4 h at 4 ℃, then
40 μL protein G beads were added for 2 h. After centrifu-
gation to discard the supernatant, the beads were washed
three times with the RIP buffer. The RNA co-precipitated
with SYNCRIP was obtained by resuspending the beads
in Trizol RNA extraction reagent and further extraction,
and then was analyzed by PCR; GAPDH served as a neg-
ative control.
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