Egfr py1068

Total cell lysates from cultured cells were used. Equal amounts (30 μg) of extracted protein were resolved on SDS-PAGE by electrophoresis, transferred and blocked in TBST (20 mM Tris–HCl, 137 mM NaCl, and 0.1% Tween 20, pH 7.5). The polyvinylidine difluoride membrane was incubated with primary antibody overnight at 4°C, and then with horseradish peroxidase conjugated secondary antibody for 1 hour. The primary antibodies used were GALNT6 (Sigma), GAPDH (Meridian Life Science), EGFR and EGFR pY1068, (Cell Signaling Technology, Inc.). Specific bands were detected by enhanced chemiluminescence detection system (Amersham, Uppsala, Sweden). Protein signals were quantified by optical density ratios using GAPDH expression as a control.

The antibodies used were obtained from the following suppliers: EGFR, EGFR-pY1068, ErbB3, ErbB3-pY1289, AKT, AKT-pS473, focal adhesion kinase (FAK), and FAK-pY397 were from Cell Signaling Technology (Danvers, MA, USA), Extracellular signal-related kinase (ERK) 1, ERK-pY204, and Integrin β1 were from Santa Cruz Biotechnology (Dallas, TX, USA), and α-tubulin was from Sigma-Aldrich.

Immunoprecipitation was performed on total protein extracted from H69 cells cultured in the presence or absence of LPS. Lysates were pre-cleared with Protein A/G sepharose beads (Santa Cruz) at 4°C and incubated with a MyD88 (Santa Cruz) or Son of sevenless homolog 1 (SOS1; Santa Cruz) antibody overnight at 4°C. Protein A/G sepharose beads were added to the samples, centrifuged, washed in ice-cold PBS, and resuspended in Laemmli sample buffer. Western blots were performed for Grb2 (Santa Cruz) or TLR4 (Imgenex). Western blots were also performed using cell lysates from untreated, siRNA or inhibitor-treated cells that were cultured in the presence or absence of LPS. Samples were run on a 4–15% gradient Tris-HCL gel, transferred to a nitrocellulose membrane, and immunoblotted with primary antibodies for Grb2 (Santa Cruz), EGFR (Cell Signaling), or EGFR pY1068 (Cell Signaling) and appropriate secondary antibody (LiCor). Membranes were visualized on the LiCor Odyssey Infrared imaging system.

B4GALNT3 proteins were detected with rabbit anti-B4GALNT3 polyclonal antibody (Sigma, St. Louis, MO). Detection of glycoproteins decorated with LacdiNAc structure was achieved by using biotinylated Wisteria floribunda agglutinin (WFA) (Vector laboratories, Burlingame, CA). For EGFR and its downstream signaling analyses, antibodies against total EGFR, EGFR pY 845, EGFR pY1068, p-AKT, AKT, p-ERK1/2, ERK1/2 (Cell Signaling Technology, Danvers, MA), and GAPDH (BD Biosciences, San Jose, CA) were used. Signals on Western blots were quantified by ImageJ 1.42q software (Wayne Rasband, NIH, Bethesda, MA).

Intestinal tissue was harvested, flushed with PBS solution and fixed as “Swiss-roll” sections in PFA for 24 h at 4 °C. For tumour scoring, intestines were fixed in Methacarn (60% methanol, 30% chloroform and 10% glacial acetic acid). Tissue was automatedly processed through the Tissue-TeK VIP infiltration Processor (Sakura) for paraffin embedding and cut into 5 µm sections with the microtome (Leica). Standard IHC techniques were conducted during this study. Antibodies used were as follows: BrdU, 1:500 (Bioss, bs-0489H), β-catenin, 1:50 (BD Biosciences, 610154), cleaved Caspase 3, 1:800 (R&D), Lyz1 (DAKO, A009), Muc2 (Genetex, GTX100664), EGFR^pY1068, 1:25 (Cell Signalling, 3777S), EGFR^pY1068, 1:400 (Abcam, ab40815) and ERK1/^2pT202/Y204, 1:100 (Cell Signalling, 4370S). At least 3 different mice of each genotype were used as biological replicates in every IHC experiment. Scoring of the staining was done blinded for evaluation and representative images were selected. Images were digitalised using the Nanozoomer Digital slide scanner (Hamamatsu) and analysed with the viewer software NDP.view2 (Hamamatsu). Scoring of tumour sections was automatedly performed using the QuPath software (qupath.github.io), whilst proliferation scoring of normal intestine was carried out manually.