bioRxiv 10.1101/783290 (2019). (Mirus Bio LLC). After 24 hours, cells were serum-starved in Dulbeccos Modified Eagles high glucose Medium (DMEM-hg) with 0.5% FBS. 24 hours later, cells were treated with 100 nM SAG (Selleck Chemicals) for another 24 hours. Firefly and luciferase activity were measured using the Dual-Luciferase? Reporter Assay Rabbit polyclonal to PBX3 System (Promega). The data analysis was performed using GraphPad Prism 7 (GraphPad Software). Results are shown as mean s.d. from 3 biologically impartial experiments. Sterol depletion assays A pcDNA3.1 vector encoding different variants of hSMO were transfected to MEFs in 6-well plates as described above. After 24 hours, the cells were treated with DMEM-hg made up of 5% lipoprotein-deficient serum (LPDS) 50, with or without 1% (w/v) hydroxypropyl–cyclodextrin (HPCD, Trappsol), 10 M sodium compactin and 50 M sodium mevalonate at 37 C for 1 hour. Then, the cells were washed by PBS and further cultured in DMEM-hg made up of 0.5% LPDS with or without 10 M sodium compactin and 50 M sodium mevalonate at BINA 37 C for an additional 24 hours. The total RNA and cDNA were prepared as described before 51. Mouse mRNA level was measured by reverse-transcription PCR (qRTCPCR) using mouse as an internal control. The primers used for qRT-PCR are transcript levels in each experimental group compared to wild-type control group were calculated using the Ct method. Results are shown as mean s.d. from three technical repeats. Comparable results were obtained in three biologically impartial experiments. Immunoblot analysis A pcDNA3.1 vector encoding different variants of hSMO were transfected to MEFs using jetPRIME Reagent (Polyplus). After 24 hours, the medium was changed to DMEM with 0.5% FCS. The cells were resuspended in RIPA buffer with 1 mM PMSF after another 24 hours. After high-speed centrifugation, the resulting supernatant was incubated with a solubilization buffer made up of 62 mM Tris-HCl pH 6.9, 15% SDS, 8M urea, 10% glycerol and 100mM dithiothreitol at 1:1 volume ratio at 37 C for 30 minutes. After electrophoresis, the proteins were transferred to nitrocellulose filters, which were then incubated with SMO antibody E-5 (1:300, Santa Cruz Biotechnology, sc-166685) at 4 C overnight, followed by the incubation of HRP-linked anti-mouse IgG (1:5000, Cell Signaling Technology) at room temperature for 1 hour. Calnexin was used as the internal control and detected by anti- calnexin (Novus #NB100C1965). Bound antibodies were visualized by a SuperSignal? West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific). The images were scanned and analyzed using an Odyssey Fc Imaging System (LI-COR Biosciences). Comparable results were obtained in three biologically impartial experiments. Extended Data Extended Data Fig. 1 Open in a separate BINA window BINA Functional characterization of the C-terminal tail of SMO in HH signaling.The domain structure of SMO is shown above. HH signaling in SMO?/? mouse embryonic fibroblasts (MEFs) transfected with pcDNA3.1 and SMO variants was measured via luciferase activity. Data are mean s.d. (n = 3 biologically impartial experiments). Extended Data Fig. 2 Open in a separate window The overall cryo-EM maps of SMOCGi complexes.a, The map of SMOCGiCSAG complex. b, The map of SMOCGiC24( em S /em ),25-EC complex. c, The map of SMOD384RCGi complex. d, The map of SMOG111C/I496CCGi complex. Each subunit is usually displayed in different colors. The 1 and 2 of CRD are labeled and the position of CRD is usually indicated by red circles. Extended Data Fig. 3 Open in a separate window Structural comparison of SMO and Gi protein in the different says.a, Structural comparison of SMOCGiCSAG complex with the previously reported structure of SMOCGiC24( em S /em ),25-EC complex (pdb: 6OT0). b, structural comparison of SMO in SMOCGiC SAG complex with inactive SMO (pdb: 5L7D). c, Structural comparison of 3.1-? SMOCGiC24( em S /em ),25-EC complex with the previously reported structure of SMOCGiC24( em S /em ),25-EC complex (pdb: 6OT0). d, Structural comparison of Gi proteins in these reported four complexes. Extended Data Fig. 4 Open in a separate window Sequence alignment of hSMO-CRD with xSMO-CRD.The cholesterol binding residues are indicated by blue circles in both of hCRD and xCRD. The secondary structures of both CRDs are indicated above.