Anti phosphotyrosine antibody 4g10

To generate a monoclonal antibody against ARHGAP33, a fragment of mouse ARHGAP33 was used as an immunogen (UNITECH, Kashiwa, Japan). Rabbit polyclonal anti-TrkB antibodies were generated using an extracellular domain of mouse TrkB (unpublished). The rabbit polyclonal anti-ARHGAP33 antibodies were described previously7 (link). Commercially available antibodies used were as follows: anti-ARHGAP33 (#HPA030118) antibody (Atlas Antibodies, Stockholm, Sweden), anti-GM130 (#610822) and anti-EEA1 (#9001964) antibodies (BD Transduction Laboratories, CA, USA), anti-PSD95 antibody (#MA1–046; Affinity Bioreagents, CO, USA), anti-TrkC (#3376) and anti-Tubulin (#2125) antibodies (Cell Signaling, MA, USA), anti-SORT1 (#ab16640) and anti-TrkB (#ab89925) antibodies (Abcam, Cambridge, UK), anti-SorLA (#sc136073) antibody (SantaCruz, CA, USA), anti-GM130 antibody (#G7295; Sigma, MO, USA), anti-phosphotyrosine (4G10) antibody (#05–1050; Millipore, MA, USA), anti-SORT1 antibody (#ANT-009; Alomone labs, Jerusalem, Israel), anti-Rab11 antibody (#71–5300; Invitrogen, MA, USA) and anti-Rab27 antibody (#18975; IBL, Gunma, Japan). All the antibodies were used at concentrations of 0.5–1 μg ml⁻¹.

AGS cells were pretreated with P. goldsteinii MTS01 (MOI 200) for 30 min followed by infection with H. pylori (MOI = 100) for additional 6 h. The cell extracts were prepared and subjected to 6% SDS-PAGE then transferred onto polyvinylidene difluoride membranes (1:1000, Millipore). The membranes were incubated with anti-phosphotyrosine (4G10) antibody (1:2000, Millipore) overnight at 4°C, and then probed with a horseradish peroxidase-conjugated secondary antibody (Millipore). The expression level of phospho-CagA was analyzed using ECL western blotting detection reagents (GE Healthcare) and recorded by AzureSpot Analysis Software with Azure 400 (Azure Biosystems).

Equal amounts of protein samples were mixed with 5× sample buffer and boiled for 5 min, separated on SDS-polyacrylamide gels, and then transferred to PVDF membrane. The membranes were blocked in 5% BSA for 1 h at room temperature, and incubated with primary antibodies overnight at 4°C. Anti-phosphotyrosine (4G10) antibody (Millipore, Billerica, MA), antibodies against C1GALT1, GAPDH, FGFR2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p-ERK1/2, anti-ERK1/2 andibodies (Cell Signaling Technology, Danvers, MA) and anti-β-actin antibody (BD Pharmingen, San Jose, CA) were used. The blots were then incubated with secondary antibody conjugated with horseradish peroxidase and immunoreacted bands were detected by ECL reagents and exposed on x-ray film.

Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose membranes (Amersham). Membranes were blocked overnight in 5% w/v BSA and incubated with primary antibody for 2 h. Anti-GST-HRP (RPN1236, 1:10000) was from Sigma-Aldrich. Anti-phosphotyrosine antibody (4G10) was obtained from Millipore. Anti-Elongin C antibody (610761, 1:3000) was from BD Biosciences. Anti-p44/42 (9102S, 1:2000), anti-STAT5 (94205; 1:3000), and anti-phospho-STAT5 (9359; 1:2000) were purchased from Cell Signaling. Anti-actin-HRP antibody (C4) was obtained from Santa Cruz (sc-47778 HRP; 1:1000). Anti-Cullin 5 (ab184177), anti-Elongin B antibody (ab154854, 1:2000), and anti-SOCS2 antibody (ab109245, 1:1000) were purchased from Abcam. Antibody binding was visualized with peroxidase-conjugated sheep antirabbit immunoglobulin (Southern Biotech; 4010-05; 1:15000), or sheep antimouse immunoglobulin (GE Healthcare; NA931-1ML; 1:10000) and the enhanced chemiluminescence (ECL) system (Amersham or Millipore).

KM12C colon carcinoma cells (Morikawa et al., 1988 (link)) were cultured in MEM (Cellgro) supplemented with 10% fetal bovine serum (FBS; Cellgro), non-essential amino acids, MEM vitamin solution and sodium pyruvate, as described previously (Espejo et al., 2010 (link)). HCT116 colon carcinoma cells were maintained in McCoy's 5A (Cellgro) medium supplemented with 10% FBS. HEK 293T cells were maintained in DMEM (Sigma) containing 10% FBS. Epidermal growth factor was purchased from Sigma. Rat-tail collagen type I was purchased from BD Biosciences. Monoclonal antibodies to p120, E-cadherin, α-catenin, p190 Rho GAP, cortactin, Living Colors™ anti-YFP antibody, JL-8, and Rac1 were purchased from BD Biosciences. Anti-E-cadherin antibody, DECMA-1, was purchased from Abcam. Mouse-specific p120 antibody, 8D11, was a generous gift from A. Reynolds (Vanderbilt University, Nashville, TN). Polyclonal rabbit anti-p120 antibody was obtained from Santa Cruz and rabbit antiserum to β-catenin was purchased from Sigma. Rabbit anti-PTPN12 (PTP-PEST) was purchased from Sigma. Anti-phosphotyrosine antibody, 4G10, was purchased from Millipore. VAV2 antibodies were used as described previously (Liu and Burridge, 2000 (link)) or purchased from Cell Signaling. Anti-phospho-Y172-VAV2 antibody was purchased from Abcam. RhoA antibody was purchased from Santa Cruz.