Comparative Studies on the In Vitro: 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).
|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|