| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | The following antibodies were used for immunoprecipitation and immunoblotting: mouse anti-Myc (9E10) (Santa Cruz, 1:5000), mouse anti-HA (Santa Cruz, 1:5000), mouse anti-Flag (M2) (Sigma, 1:5000), mouse anti-β-actin (GenScript, 1:10000), rabbit anti-PKAα (Santa Cruz, 1:500), rabbit anti-PKAIβ (Santa Cruz, 1:500), goat anti-rabbit HRP (1:10000; Jackson ImmunoResearch) and goat anti-mouse HRP (1:10000; Jackson ImmunoResearch). For RNA interference experiment, dsRNA was generated through in vitro transcription by using the MEGAscript T7 kit (Ambion). Standard RT-qPCR primers for Drosophila mRNA-cap (upstream: 5′-AAAAACCATTTAAAGCGC-3′ and downstream 5′-GCTTTGCGCGATCACTAGC-3′), ci (upstream: 5′-CCTCTTGCGTATTCTGAATT-3′ and downstream: 5′-GAATCTGATGTTCCACCCGT-3′), smo (upstream: 5′-CAGCTATACAGCCCTTTTTG-3′ and downstream: 5′-CACTGGCCAGTTCCGTTGAA-3′), PKA-C1 (upstream: 5′-CACGAAAGACTATTATGCC-3′ and downstream: 5′-CGCGTAGAAGCGCGAGTGCGGCTCCG-3′), PKA-C3 (upstream: 5′-GCCGCTCGAACGGCCGAAG-3′ and downstream: 5′-GGTGGTGGTGGTGGTGGCGG-3′), PKA-R1 (upstream: 5′-GCGTTCTGAGCAGGGCGAGG-3′ and downstream: 5′-CCCAACACGCGTTCAAATC-3′), PKA-R2 (upstream: 5′-ATATGCCCAGAGCGGCCACCGTGC-3′ and downstream: 5′-GGCGGC GTCACCCTGTTTGATG-3′) and actin (upstream: 5′-GTACCCCATTGAGCACGGTA-3′ and downstream: 5′-CGAACATGATCTGGGCATC-3′) were synthesized by GenScript. | Get A Quote |
Hedgehog (Hh) signaling plays a pivotal role in animal development and its deregulation in humans causes birth defects and several types of cancer. Protein Kinase A (PKA) modulates Hh signaling activity through phosphorylating the transcription factor Cubitus interruptus (Ci) and G protein coupled receptor (GPCR) family protein Smoothened (Smo) in Drosophila， but how PKA activity is regulated remains elusive. Here， we identify a novel regulator of the Hh pathway， the capping-enzyme mRNA-cap， which positively regulates Hh signaling activity through modulating PKA activity. We provide genetic and biochemical evidence that mRNA-cap inhibits PKA kinase activity to promote Hh signaling. Interestingly， regu... More
