Phospho ERK, p38 MAP kinase anti-anti-phospho-p38 MAP kinase and anti-phospho-ErbB3 antibody Body were from Cell Signaling Technology. A potent inhibitor of PI 3-kinase was a kind gift from Dr. ZSTK474 S. Yaguchi. A MEK inhibitor, PD98059, and inhibitor of p38 MAP kinase, SB202190, GSK1349572 S/GSK1349572 were obtained from Wako Co. Ltd. Ltlich. We have SB203580 9 is used as an inhibitor of p38 MAP kinase. However, the results were always the same as those with SB202190. We pr Sentieren therefore the results of SB202190 in this document. SecinH3 was from Millipore. Immunocytochemistry, cells were fixed with 3.7% formaldehyde. After treatment with PBS with or without Triton X100 0.2%, the cells with primary Ren Antique Rpern were incubated for 1 h and visualized with Alexa488 or Alexa594 conjugated second antibody Body.
F-actin was of TRITC phallocentrism Dine visualized. The cells were observed by confocal microscopy. Trichostatin A Western blot Immunpr Zipitation and Western biotinylation of cells was performed using a PVDF membrane, as described above. Immunpr Zipitation and biotinylation cells were performed as previously described. Fractionation of cells in a buffer that were exposed to 10 mM Tris-HCl, pH 7.5, 10 mM NaCl and proteinase inhibitor cocktail, they were then homogenized in a Dounce homogenizer. After removal of the nuclei by centrifugation at low speed, the cytoplasmic fraction was further fractionated into the membrane and cytosolic fractions min by centrifugation at 45,000 rpm for 60 min. The investigation of cell growth 16 105 cells were plated in 3.5 cm. The cells were harvested by trypsinization and every day.
Flow cytometry After fixation in 3.7% formaldehyde and transferred the cells were scraped off although a shot to break cell aggregates. After treatment with anti-MUC1 antibody Body, the cells were washed once with PBS and reacted with a second antique Body conjugated Alexa488, no additionally USEFUL activation not detected not detected HSC45, HSC58 activates no reaction, no additionally USEFUL activation HSC60 is no response to NUGC4, no additionally USEFUL activation is enabled KatoIII are not additionally USEFUL activation aStatus ErbB2 and ErbB3, HRG and responses in different cell lines Signet carcinoma presents pr. HSC45, HSC58, HSC60 and CRO In the form of mixtures with other cell types, and can not be purified. doi: 10.1371/journal.pone.0029599.
t001 Figure 1 Response of cells to stimulation HRG HCC2998. HCC2998 cells were treated with HRG for the indicated time. The cells were microscopically. Observed, SB 16 h, indicating SB202190 more for the culture after incubation with HRG for 16 hours. HCC2998 cells were treated with HRG for the indicated time. Protein phosphorylation and steps were analyzed by Western blot. HCC2998 cells were treated with HRG in the presence of PD98059, SB202190 or ZSTK474 for 24 h. The cells were observed under a microscope. doi: 10.1371/journal.pone.0029599.g001 heregulin stimulation of MKN45 cells HCC2998 and a second December 2011 | Volume 6 | Issue 12 | e29599 Healthcare. After washing with PBS three times, the samples were subjected to flow cytometry. Results HCC2998 cells express ErbB2 and ErbB3 and respond to HRG HCC2998 cells were treated withHRG. As shown in Fig. 1A, was the loss of contact with cells of a few hours after HRG stimulation was observed. Since the cells do not move much, it was hard to tell