cipitation and western blot analysis were performed using standard protocols as previously described [20, 30]. The antibodies used were mouse anti-Myc (Abmart); mouse anti-Flag, M2 (Sigma); mouse anti-HA, F7 (Santa Cruz); and rat anti-Ci, 2A [45]. To induce Fu dimerization based on FKBP, the indicated constructs were transfected into S2 cells. At 10 or 20 h for phosphorylation and luciferase assays before harvested, respectively, cells were treated with or without AP20187 (50 nM; kindly provided by ARIAD Pharmaceuticals). In gradient treatment assay, the con- centration of AP20187 changed from 0 nM to 50 nM. Leptomycin B (LMB, Calbiochem) was used at 5 nM to treat transfected cells for 2 h before cells were harvested. In Ci155 dissociation assay, to inhibit Ci155 degradation, MG132 (Sigma) was applied at 50 μM to treat cells for 6 h before cells were harvested. Immunoprecipitation based kinase assay S2 cells were transfected with indicated Fv2- or Div-based Fu constructs, Flag-tagged Cos2 or Su(fu), respectively. Cells were then harvested and treated with standard immunoprecipitation worksheet, respectively. The beads were washed with 1 ml kinase assay buffer for three times at 4 ºC, 10 min for each time. The im- munoprecipitated products for FKBP-Fu and Cos2 or Su(fu) were mixed with 50 μM ATP and 100 nM AP20187 in kinase buffer, and then incubated at 30 ºC for 60 min. For Div-Fu phosphoryla- tion assay, immunoprecipitated products for Div-Fu and Cos2 or Su(fu) were mixed with 50 μM ATP in kinase buffer, and then in- cubated at 30 ºC for 60 min. After the reaction, the samples would be treated for western blot following the standard protocol. The kinase assay buffer consists of 20 mM Tris-HCl, pH 7.5; 20 mM β-glycerophosphate; 10 mM MgCl2; 50 mM NaCl; 100 μM ortho- vanadate; 0.5 mM NaF; 0.1 mM Na3VO4 and proteinase inhibitor (Sigma Cop.). For conforming that the mobility shift was caused by the protein phosphorylation, the phosphatase (Fermentas Cop.) was used in the kinase assay. RNAi, immunostaining, luciferase reporter assay and FRET analysis The double-strand RNA was synthesized using the in vitro Transcription T7 Kit from TakaRa. After cells were transfected for 24 h, the culture medium was changed to Serum Free Medium with 20-50 μg dsRNA/106 cells for 1-6 h starvation. Then fresh medium with serum was added and cells were cultured for 24-36 h. The targeting amino acids and primer sequences are as follows: Fu, targeting amino acids 302-525, Fu-RNAi-F, 5′-TAATAC- GACTCACTATAGGGAGATTTGGCGAGTCGCGACAGG-3′, Fu-RNAi-R, 5′-TAATACGACTCACTATAGGGAGATGT- TCAAGAAGGCTAGCCACTC-3′; Cos2, targeting amino acids 621-841, Cos-RNAi-F, 5′-TAATACGACTCACTATAGGGAGA- CAGCCGCCTCCGCCCATCGAT-3′, Cos-RNAi-R, 5′-TAATAC- GACTCACTATAGGGAGACGTAATCCTAGCCTGTACTGC-3′; GFP, targeting amino acids 2-201, GFP-RNAi-F, 5′-TAATAC- GACTCACTATAGGGAGAGTGAGCAAGGGCGAGGAGC-3′, GFP-RNAi-R, 5′-TAATACGACTCACTATAGGGAGAGG- TAGTGGTTGTCGGGCAGC-3′; Renilla-RNAi-F, 5′-GATCAC- TAATACGACTCACTATAGGGATGACTTCGAAAGTTTAT- GATCCAG-3′, Renilla-RNAi-R, 5′-GATCACTAATACGACT- CACTATAGGGTTATCTTGATGCTCATAGCTATAATG-3′; Smo, targeting 201-862bp of Smo cDNA, Smo-RNAi-F, 5′-TTAATAC- GACTCACTATAGGGAGACAGGGACGACAAACCCTG- www.cell-research.com | Cell Research Yanyan Zhang et al. npg 1449 GTTTG-3′, Smo-RNAi-R, 5′-TTAATACGACTCACTATAGGGA- GAGATACTTGTTGGCATTCTTCC-3′ [13]. Cell staining was carried out as described [9]. Imaginal dick immunostaining was performed as described [12]. Primary antibodies used in this study were mouse anti-Myc (Abmart); mouse anti-Flag, M2 (Sigma); mouse anti-GFP 1:100 (Abmart); rat anti-Ci, A2[45]; rabbit anti- βGal (Cappel). The Ci-responsive ptc-Luc reporter was described earlier [17]. In all luciferase assays, 1 × 106 S2 cells were trans- fected with 300 ng ptc-luc reporter, 6 ng Renilla, 300 ng Gal4, 100 ng Ci155, 100 ng Su(fu) and 300 ng indicated constructs in each well of a 24-well plate. GFP RNAi was used as control in the indicated luciferase assay. The luciferase activity was tested after 48 h using the dual-reporter luciferase system on a 96 lumi- nometer (Promega). The normalized data are expressed as mean of at least five replicates obtained from at least three independent experiments. For FRET analysis of cultured cells, CFP- and YFP- tagged constructs were transfected into S2 cells together with an ub-Gal4 expression vector. Transfected cells were treated with or without Hh conditional medium. For maximal Hh signal strength, a UAS-Hh expression construct was also included in the transfec- tion. Cells were washed with PBS, fixed with 4% formaldehyde for 20 min, and mounted on slides in 80% glycerol. Each data set was based on 15-20 individual cells. In each cell, three to five re- gions of interest in photobleached area were selected for analysis. In the absence of Hh, only limited Smo colocalizes with Cos2/ Fu complex. Under this condition, only areas, in which Smo co- localized with Cos2/Fu complex, were selected for collecting FRET signal. For FRET assay of wing discs, transgenes were ex- pressed with MS1096 for analysis of A or P compartment and A/P boundary cells. Late third instar larvae wing discs were dissected in PBS, fixed in 4% formaldehyde and mounted on slides in 80% glycerol. Each data set was based on 5-10 individual discs. For collecting FRET change information, Leica LAS SP5 confocal mi- croscope was employed. CFP was excited at 458 nm wavelength and the emission was collected through a BP 480-520 filter. YFP was excited at 514 nm wavelength and the emission was collected through a BP 535-590 nm filter. CFP signal was obtained once before (BP) and after (AP) photobleaching YFP using the full power of 514 nm laser line for 1-2 min at the top half of each cell or selected dick area, leaving the bottom half as the internal con- trol. The intensity change of CFP was analyzed using the ImageJ software (NIH). The efficiency of FRET was calculated using the formula: FRET% = [(CFPAP−CFPBP)/CFPAP] × 100. Real-time PCR assay We used real-time PCR to identify the RNAi efficiency for Cos2, Fu and Smo because of the absence of antibodies for en- dogenous protein. In this assay, S2 cells treated by the indicated Cos2, Fu or Smo dsRNA or control Renilla dsRNA were harvested and used to prepare the total RNA using standard RNA isolation protocol. Then the RNA products were used to synthesize the first strand cDNA using the Kit (#K1622) from Fermentas Company. Finally, the real-time PCR was done using the SYBR green mas- ter mix (QPK-201) from TOYOBO on the instrument of ABI- 7500 (Applied Biosystem Cop.). The target regions of genes in the real-time PCR were different from the RNAi target regions. The primers used in this assay were tested to have good specificity and proliferation rate. The 2-∆∆Ct method was used for the relative quantification [46]. Rp49 was used as the internal control for the