For in vitro kinase assay, GST-SmoK containing aa656–753 of Smo, His-Krz, and His-Krz∆R fusion proteins were expressed in bacteria and purified with GST beads. 3 μg of Smo were incubated at 30 °C for 30 min in 50 μL of assay buffer (20 mM Tris-HCl at pH8.0, 10 mM MgCl2, 0.2 mM EDTA, 1 mM DTT), and 2.5 μM ATP in the presence of commercial recombinant PKA and CK1 (New England Biolabs) followed by western blotting with antibodies to examine Smo phosphorylation. Antibody used: rabbit anti-GST (Santa Cruz, 1:500), anti-SmoP (1:20)16 (link), mouse anti-His (H8, Millipore, 1:1,000).
To examine the levels of gene expression, RT-PCR was carried out using S2 cells with the primers for Ubc9 (5′-TGG CGC AAG GAT CAC-3′; 5′-GCC CGC CCT CCC AGG-3′), PIAS (5′-CAG CTG CCT AAT GTC ATT C-3′; 5′-GAC ACC ACT GAA CCG-3′), Smt3 (5′-AGA AGG GAG GTG AGA C-3′; 5′-CGT TCA TCA GCT TCC TC-3′), and Ulp1 (5′-CGG GAT TCC AGG CTC-3′; 5′-GTC CAC ACG CCG GTA C-3′).
The ptc-luc reporter assay has been described with S2 cells cultured in 6-well plates and transfected with 50 ng tub-Ci and 150 ng ptc-luc reporter constructs followed by activity analysis using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) combined with the GLOMAX Multi Detection System (Promega)16 (link). Each ptc-luc experiment was repeated three times and the error bars indicated standard deviation (S.D.) from four repeats.
To examine the levels of gene expression, RT-PCR was carried out using S2 cells with the primers for Ubc9 (5′-TGG CGC AAG GAT CAC-3′; 5′-GCC CGC CCT CCC AGG-3′), PIAS (5′-CAG CTG CCT AAT GTC ATT C-3′; 5′-GAC ACC ACT GAA CCG-3′), Smt3 (5′-AGA AGG GAG GTG AGA C-3′; 5′-CGT TCA TCA GCT TCC TC-3′), and Ulp1 (5′-CGG GAT TCC AGG CTC-3′; 5′-GTC CAC ACG CCG GTA C-3′).
The ptc-luc reporter assay has been described with S2 cells cultured in 6-well plates and transfected with 50 ng tub-Ci and 150 ng ptc-luc reporter constructs followed by activity analysis using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) combined with the GLOMAX Multi Detection System (Promega)16 (link). Each ptc-luc experiment was repeated three times and the error bars indicated standard deviation (S.D.) from four repeats.
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