For caspase-3 cleavage, treated cells had been lysed working with RIPA and analyzed by SDS-PAGE followed by Western blotting implementing an antibody exact for cleaved caspase-3 . MDA-MB-231 Xenografts in Nude Mice NCR-nude female athymic mice have been bought from Taconic Farms, Inc. Mice were injected during the flank area with one.56106 MDA-MB-231 cells, though anesthetized with ketamine and xylazine. Prior to treatment, tumors were measured in 3 dimensions utilizing a caliper and tumor volume was calculated by multiplying the 3 measured dimensions by 0.5 . When the tumors reached a minimum size of one hundred mm3, mice have been injected intraperitoneally, twice per week, with 200 mL in the TE- 64562 peptide , Tat peptide in PBS or PBS. Endpoints have been defined because the tumor dimension reaching 2000 mm3 or 20 mm in any dimension, vital excess weight loss taking place or in case the mouse appearing unhealthy according to entire body conditioning scoring requirements.
For histological evaluation, organs have been collected post-sacrifice and fixed in 4% U0126 formaldehyde in PBS, followed by paraffin embedding. Sections have been mounted onto positively charged slides and H&E stained. Images had been captured at 206resolution on a Zeiss Axioplan 2 microscope. EGFR-peptide Biotin-binding Assay The EGFR constructs had been obtained from the laboratory of Dr. Graham Carpenter of Vanderbilt University and described previously . SK-N-MC cells had been plated in 10-cm dishes, grown to 90% confluence, transfected with the indicated EGFR construct making use of Lipofectamine 2000 for 6 hours and serum starved overnight. The cells have been treated with the biotinylated peptides for 2 hours, washed with phosphate buffer containing 500 mM NaCl pH 7.4, and then lysed by sonication in immunoprecipitation buffer .
The lysates were incubated with prewashed, streptavidin-coated beads overnight at 4uC with rotation. For the reverse experiment, biotinylated peptides have been incubated with streptavidin-coated beads for 2 hours at 4uC with rotation and washed. Transfected SK-N-MC cells were lysed, as above and incubated with the peptide-conjugated beads overnight at 4uC with rotation. Shikimate The resulting bead-precipitates have been washed and analyzed by Western blot with the indicated antibodies following standard procedures and visualized by chemiluminescence. Cell Therapy and Lysate Collection for Western Blot Evaluation Cells have been plated in 10-cm dishes, grown to 80% confluence, serum starved overnight, treated and analyzed.
For dose response assays, the peptides, EGFR-specific tyrosine kinase inhibitor or control was added to each plate and incubated at 37uC for 30 minutes, followed by EGF for an additional 10 minutes at 37uC. For timecourses, TE-64562 and/or EGF had been added and the cells had been incubated at 37uC for the indicated amounts of time. For EGFR dimer detection, cells had been crosslinked after treatment method applying BS3 crosslinker according to the manufacturers instructions.