Goat anti rabbit antibody

To characterize mutated Cspg4, HEK293T cells were seeded at 4 × 10⁵ cells/ 6 cm² dish and cultured using 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA) Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA). After cultured for 24 hours, culture medium was change to FBS free DMEM and transfected with pT7-cspg4^stop-FLAG-IRES-dsRed or mutated pT7-mcspg4^stop-FLAG-IRES-dsRed by using TransIT-X2 Transfection Reagent (Mirus Bio, Madison, WI, USA). Twenty four hours after transfection, culture medium was collected and concentrated by Amicon Ultra-15 30K (Millipore Co., Billerica, MA, USA) according to manufacturer’s instruction. The total protein of cells was extracted with lysis buffer (50 mM Tris HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, and 1% TRITON X-100). The BCA protein assay kit was used to quantify the protein concentration. Sixty or 20 μg total protein from culture medium or cell lysate was separate by 8% SDS-polyacrylamide gel electrophoresis (PAGE) and subsequently transfer onto a PVDF membrane. The membrane was blocked and probed with a 1:2000 dilution of DDDDK tag antibody (ab21536, Abcam, Cambridge, MA, USA), and a 1:5000 dilution of goat anti-rabbit antibody (PerkinElmer Inc., Waltham, MA, USA) was used as secondary antibody. The signal was detected by Chemiluminescence Reagent (Immobilon Western, Millipore Co., Billerica, MA, USA).

To investigate the interaction between Cspg4 and Wnt11, HEK-293T cells were transfected with pT7-cspg4^stop-FLAG-IRES-dsRed or pT7- wnt11^stop-HA-IRES-dsRed, or co-transfected with both plasmids as above described. After total protein was extracted by lysis buffer, Anti-FLAG M2 Magnetic Beads (Sigma-Aldrich) were used to precipitate flag-tagged Cspg4 protein according to the manufacturer’s instruction. After immunoprecipitation, samples were separated by 12% SDS-PAGE or 3~8% tris-acetate gel for detecting HA-tagged Wnt11 or flag-tagged Cspg4, respectively. Protein was transfer onto PVDF membranes and blocking with 5% bovine serum albumin (Sigma-Aldrich). After blocking, membrane was hybridized with HA-Tag (C29F4) Rabbit mAb (#3724, Cell Signaling Technology, Danvers, MA, USA) or DDDDK tag antibody (ab21536, Abcam), and a 1:5000 dilution of goat anti-rabbit antibody (PerkinElmer Inc.) was used as secondary antibody. The signal was detected by Chemiluminescence Reagent.