Anti myc antibody 9e10

Cells were lysed with RIPA-Doc buffer (Tris-HCl, 50 mM; NaCl, 150 mM; Triton X-100, 1%; deoxycholate, 1%; and SDS, 0.1%; supplemented with 1/100 protease inhibitors). Cell lysates were separated by 10% Tris-glycine SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked in 5% nonfat milk for 1 h, then incubated overnight at 4°C with anti-myc (9E10), anti-GFP, anti-RCAN1, and anti-β-actin (AC-15) antibodies, respectively [23 (link)]. The membranes were washed in TBST with 0.1% Tween-20 and incubated with HRP-labeled goat anti-mouse or HRP-labeled goat anti-rabbit antibodies at room temperature for 1 h. Anti-myc antibody 9E10 was obtained from Abcam. β-Actin antibody AC-15, HRP-labeled goat anti-rabbit antibody, and HRP-labeled goat anti-mouse antibody were obtained from ZSGB-BIO. Anti-GFP antibody was purchased from Proteintech Group. Anti-RCAN1 antibody was purchased from Sigma. The image was obtained by using FluorChem R imaging system.

We prepared wild-type and domain-deleted hMuSKect-myc, as well as hLRP4-FLAG and FLAG-CTD for in vitro plate-binding assays and the ATF2-luciferase reporter assay, as described previously38 (link). Wild-type or mutant phMUSKect-myc, or phLRP4N-FLAG or pFLAG-CTD, was transfected into HEK293 cells in a 10-cm dish using the calcium phosphate method44 (link). We purified hMuSKect-myc with the c-myc-Tagged Protein Mild Purification Kit version 2 (MBL). We also purified hLRP4N-FLAG and FLAG-CTD with the Anti-DYKDDDDK-tag Antibody Beads (Wako). We confirmed the presence of isolated hMuSKect-myc by Western blotting with anti-myc antibody (9E10, Abcam), and hLRP4N-FLAG and FLAG-CTD with anti-FLAG antibody (M2, Sigma-Aldrich). We also confirmed the purity of isolated recombinant proteins by SDS-PAGE followed by protein staining with the Oriole Fluorescent Gel Stain (Bio-Rad).