Hepatitis B Virus (HBV)

Comparative Studies on the In Vitro: MATERIALS AND METHODS(5)

MATERIALS AND METHODS(5)

Cells were lysed with TriReagent (Molecular Research Center, Cincinnati, OH), and RNA and protein were extracted using the protocol provided by the manufacturer. Cellular protein content was measured using the Micro BCA protein assay kit (Pierce, Rockford, IL).

Immunoblotting for aSMA

Proteins (7.5 ^g) from cell extracts were separated by one-dimensional SDS-PAGE (10%). The proteins were then transferred to nitrocellulose membranes (MSI, Westboro, MA) and incubated with an aSMA monoclonal antibody (Dako) at a 1:1000 dilution. Horseradish peroxidase-conjugated second antibody was used at a 1:3000 dilution, and the immunoreactive product was visualized using the enhanced chemiluminescence system (Pierce). Myometrial tissue extracts were included as positive controls for aSMA on all Western blots. Specificity of aSMA induction was confirmed by using two other monoclonal antibodies against actin (kindly provided by Dr. James Lessard, Children’s Hospital Research Foundation, Cincinnati, OH. buy flovent inhaler

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

The RNA isolated from cells was reverse transcribed, and the cDNA was subjected to PCR. As a control, an RNA preparation that was not reversed transcribed was also subjected to PCR to ensure that no genomic contamination was being amplified. For the detection of different cDNAs, primers were designed from nucleotide sequences provided by GenBank (Table 1).

TABLE 1.

Protein Primer sequence Product size (bp) GenBank accession num
aSMA sense: GGA ATG GGA CAA AAA GAC AG antisense: CAG CAG TAG TAA CGA AGG AA 477 X13839
IGFBP-1 sense: TGC TGC AGA GGC AGG GAG CCC antisense: AGG GAT CCT CTT CCC ATT CCA 379 M31145
H3.3 sense: GCA AGA GTG CGC CCT CTA CTG antisense: GGC CTC ACT TGC CTC CTG CAA 213 Reference
RIa sense: AAG ACA ATG GCC GCT TTA GC antisense: CCC AAC ACT GGT TGC CCA TT 217 M18468
RIP sense: TCC CCG GAC TCA CTT TTT GG antisense: ATC TGG GGG ACC TGA CCT CA 267 M65066
Rlla sense: GAA CGA ATG AAG ATT GTG GA antisense: CTT CAT GAT GTC CAT GCA GG 333 X14968
RIIP sense: TGT GCA GAA GCT TAT AAT CC antisense: AGG AGA GGT AGC AGT GAT TG 378 M31158

Category: Decidualization Process

Tags: Decidualization Process, Human, Papio anubis

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