Template cDNAs were obtained by reverse tran scription of total RNAs using oligo primer and superscript II reverse transcriptase. Amplification was carried out using SYBR Green qPCR Master Mix. Sequences of the real time qRT PCR primers used are listed in Table 1. Quantitative real time qRT PCR was performed using an ABI System Sequence Detector 7300 under the following ther mocycler conditions selleck compound stage 1, 95 C for 30s, 1 cycle. stage 2 95 C for 5 s and 60 C for 31 s, 40 cycles. B actin was used as an internal control for the expression levels of target genes. Relative mRNA levels were calculated in terms of the average cycle number of PCR amplification was used to calculate the amount of RNA relative to the control. In situ hybridization In situ hybridization was performed as previously described.
The human cDNA vectors for WNT2, WNT7B, FZD7, LRP6, DVL2, DLV3, GSK 3B, APC, AXIN2 and TCF4 digoxygenin labelled probes were amplified Inhibitors,Modulators,Libraries from cDNA templates, which were synthesized from total RNA derived from human lung at 17 W. Sequences of the PCR primers and the enzyme sites used for insertion into the pBluescriptII KS plasmid are shown in Table 2. Other digoxygenin labelled RNA probes for in situ hybridization of lung sections were generated from the following tem plates FZD4, LRP5 and LEF1 plasmids, CTNNB1 and SOSTDC1 plasmids. Tissue sections hybridized with sense probes were used as nega tive controls. Pseudoglandular explant cultures and CHIR 99021 treatment Pseudoglandular explant cultures were prepared as previ ously described.
Briefly, fetal human lungs were isolated from surrounding structures, cut into cubes and divided into four groups. One group was dehydrated in a graded ethanol series and embedded in paraffin. The remaining three groups were cultured on an air liquid interface using permeable supports in Inhibitors,Modulators,Libraries DMEM media in the absence or presence of CHIR 99021. Explants were cultured at 37 C in 95 % air/5 % CO2 for up to 72 h prior to collection for analyses. A stock solution of CHIR 99021 was prepared at 1 mM and stored at 4 C. Western blotting Protein extracts of human lung explants before or after Inhibitors,Modulators,Libraries CHIR 99021 treatment for 72 h were prepared according to a previously described nuclear protein ex traction protocol. Extracts were subjected to West ern blot analysis using mouse anti human B CATENIN and mouse anti human B actin primary antibodies.
Blots Inhibitors,Modulators,Libraries incubated in the absence of the primary antibodies were used as negative controls. The Polink 2 plus Polymer HRP Detection Sys tem for mouse primary antibodies was used as the secondary Inhibitors,Modulators,Libraries antibody. Western Blotting Chemilumins cence Luminol Reagent was added to detect immunopositive protein bands. The data was obtained from triplicates of each independent experiment. sellckchem Values were normalized to corresponding B ACTIN levels and then expressed as a percentage of the control value.