Anti perk antibodies

Tissue specimens were fixed in 10% neutralized formalin overnight, preserved in 70% ethanol, and then embedded in paraffin. Tumour diagnosis on haematoxylin and eosin-stained sections was performed by two veterinary pathologists (Y.K. and L.B.) blinded to the genotype or treatment. Immunohistochemistry staining was performed on tissue sections using anti-mouse CD3 (Thermo Scientific, clone: SP7, 1–200 dilution)49 (link) or anti-pErk antibodies (Cell Signaling Technology, 1–500 dilution)50 (link). The percentage of the lung with disease, the percentage of lung tumours positive for pErK, and the grade of the lung tumours were assessed without knowledge of genotype by a single observer (E.J.M.) following protocols described previously51 (link) with a slight modification. Lung tumours were graded as low-, intermediate- or high-grade. Low-grade tumours formed a solid tumor and displayed regular to slightly irregular nuclei. Intermediate-grade tumours had enlarged, pleomorphic nuclei with prominent nucleoli and nuclear moudling. High-grade tumours displayed all of the characteristics of intermediate-grade lung tumours in addition to aberrant mitoses, tumour giant cells and desmoplasia.

Antibodies specific for MEKK1 were purchased from Santa Cruz Biotechnology. Anti-pERK antibodies were purchased from Cell Signaling Technology. U0126 was purchased from Promega (Madison, WI), CAY10512 was purchased from Cayman Chemical (Ann Arbor, MI), SSR128129E was purchased from Selleckchem (Houston, TX), Maraviroc, and PEITC were purchased from Sigma-Aldrich (St. Louis, MO).

Immunohistochemistry was performed according to our previously published procedures [62] (link), [63] (link). The following antibodies were used in this study: rabbit polyclonal anti-β-galactosidase antibody (1∶100, Cappel), Guinea pig polyclonal anti-Piwi antibodies (1∶100; produced by H. Lin), chicken polyclonal anti-GFP antibody (1∶200, Jax), mouse monoclonal anti-Hts antibody (1∶50, DSHB), mouse monoclonal anti-Yb antibody (1∶200; kindly provided by Dr. H. Siomi), rabbit polyclonal anti-pS137 H2Av antibody (1∶100, Rockland), rabbit monoclonal anti-pS423/425 Smad3 antibody (1∶100, Epitomics), rabbit polyclonal anti-pERK antibodies (1∶25, Cell Signaling) and rat monoclonal anti-Vasa antibody (1∶50, DSHB). All images were taken with a Leica TCS SP5 confocal microscope.

Cells were collected and lysed in RIPA (Yeasen, Shanghai, China) with protease inhibitor cocktail (Sigma-Aldrich, St.Louis, MO, USA). Equivalent protein was loaded onto 10% SDS-PAGE gel and transferred into nitrocellulose membranes (PALL, NewYork, USA). The blots were incubated with specific primary antibodies, followed by secondary antibodies. The proteins were visualized with the Clinx ChemiScope (Clinx Science Instruments, Shanghai, China). The antibodies used for western blot were listed as follows: anti-AKT antibodies (#9272, Cell Signaling Technology), anti-p-AKT antibodies (#4060, Cell Signaling Technology), anti-mTOR antibodies (#2983, Cell Signaling Technology), anti-p-mTOR antibodies (#5536, Cell Signaling Technology), anti-ERK antibodies (#4695, Cell Signaling Technology), anti-p-ERK antibodies (#4370, Cell Signaling Technology), anti-GAPDH antibodies (#5174, Cell Signaling Technology).

Leaves detached from 10–day–old rice or 2–week–old wheat seedlings were treated with 10 μm OsPep plus 0.05% silwet L–77 or silwet L–77 only (mock) for 15 or 30 min. Treated leaves were ground in liquid nitrogen, and the tissue powder was immediately mixed with the 6 × SDS–PAGE loading buffer and heated at 95 °C for 5 min. Total proteins were resolved in a 10% SDS–PAGE, and MAPK activation was evaluated by immunoblotting using anti–pERK antibodies (Cell Signaling Technology, Beverly, MA, USA).