Mouse anti 6x his

The same volume of SDS sample buffer (100 mM Tris-HCl, 200 mM dithiothreitol, 4 % (w/v) SDS, 0.2 % (w/v) bromophenol blue, 20 % (v/v) glycerol, pH 6.8) was added to the protein samples and heated for 10 min at 95 °C. The proteins were separated by SDS PAGE and transferred to a polyvinylidene fluoride membrane using semidry electroblotting (Trans-Blot SD, Bio-Rad, Hercules, CA, USA). Afterwards, the membrane was blocked with 5% BSA in PBS over night at 4 °C under shaking. The immunodetection was conducted using first a His₆ antibody (1:1000 in PBS with 0.01% NaN3, 1 h at RT, mouse anti-6x-His, Thermo Fisher Scientific, Waltham, MA, USA), followed by a horseradish peroxidase (HRP) coupled antibody (1:6000 in PBS with 0.1% Tween20, 1.5 h at RT, rabbit anti-mouse, Thermo Fisher Scientific, Waltham, USA). The washed membrane was treated with Immuno Cruz Western Blot Luminol Reagent (Santa Cruz Biotechnology, Dallas, TX, USA) and the luminicence was detected by chemiluminescence reader (ChemoCam ECL imager, Intas, Göttingen, Germany).

All reagents were from Sigma-Aldrich unless otherwise noted. The mouse anti-MUC1-ED (catalog number MA1-06503), hamster anti-MUC1-CD (catalog number MA5-11202), rabbit anti-FLAG (catalog number 740001), mouse anti-6XHis (catalog number 37-2900), horseradish peroxidase (HRP)-conjugated rabbit antimouse IgG (catalog number 31450), HRP-conjugated goat-anti-rabbit IgG (catalog number 31460), and HRP-conjugated goat antihamster IgG (catalog number PA1-28823) antibodies were from Thermo Fisher Scientific. The rabbit antibodies to NEU1 (catalog number ab233119), PPCA (catalog number ab217857), and c-Met (catalog number ab216574); and the mouse antibodies to PI3K (p85) (catalog number ab86714), p53 (catalog number ab154036), PDGFRβ (catalog number ab69506), Akt (catalog number ab108202) and phosphorylated Akt (catalog number ab105731) were from Abcam. The mouse antibodies to c-Src (catalog number 612378), EGFR (catalog number 610017), γ-catenin (catalog number 610253), and GST (catalog number 610719) were from BD Biosciences. Glutathione-agarose and Ni-NTA-agarose were from GE Healthcare Bio-Sciences. Biotinylated PNA and HRP-conjugated streptavidin were from Vector Laboratories.