Ure S3 Western blot of PK-resistant fragments. In unpurified (1) and purified GPI- PrPSc (2). Both buy Argipressin Samples were digested with proteinase K, 25 mg/ml and 10 mg/ml, respectively, treated with PNGase F and resolved on a Tricine-SDS-PAGE gel. WB was probed with the R1 antibody. (TIF) Figure S4 Bayesian protein reconstruction of the nanoLC-ESI-MS spectra of PK-treated purified GPI2 PrPSc. The mass graphs of the three peaks: 17148 Da (top), 16729 Da (middle) and 16371 Da (bottom), identified by ESI-TOF are shown. (TIF) Figure S5 Western blot of recombinant MoPrP(23?31)cleavage by PK. Samples were digested with different concentrations of PK: 0, 0.2, 1, 5, 10 and 25 mg/ml. Samples were subjected to Tricine-SDS-PAGE and the blot was probed with R1 antibody. (TIF)Figure S6 Schematic representations of the data. A. A scheme of GPI2 PrP sequence, showing the PK-resistant areas (blue squares) and the PK cleavage points and flexible areas (gray line). B. Lengthwise comparison of the different peptides found by limited proteolysis and MALDI-TOF analysis (colors match those displayed in Figure 2). (TIF)Acknowledgments ImmunohistochemistryImmediately after extraction, the brain was fixed in formalin and then sliced into four transversal sections by cutting the brain caudally and rostrally to the midbrain and at the level of the basal nuclei. The sections were dehydrated by equilibration in solutions of progressively higher ethanol concentration and then equilibrated with xylene before being embedded in paraffin. Haematoxylineosin was used to stain the 4 mm thick sections. Additional sections were mounted on 3-triethoxysilyl-propylamine-coated glass slides for immunohistochemical (IHC) studies. These brain sections were deparaffinised, immersed in formic acid containing peroxidase inhibitors, and autoclaved prior to IHC analysis. These autoclaved samples were washed, treatedWe thank Bruce Chesebro, Rocky Mountain Laboratory, NIH, MT, USA, for his kind gift of GPI- mice, Hanna Serban, Institute for Neurodegenerative Diseases, UCSF, CA, USA, for generously providing antibody R1, ?Juan Maria Torres, CISA, Madrid, Spain, for RML inoculum, Valerie Sim, University of Alberta, Edmonton, Canada, for advice on GPI- PrPSc Felypressin web isolation and Melissa L. Erickson, USDA, for help in preparing the manuscript.Author ContributionsConceived and designed the experiments: EVF JA CJS JRR. Performed the experiments: EVF JA EV ID. Analyzed the data: EVF JA EV CJS JRR. Contributed reagents/materials/analysis tools: MAP AR LS BP. Wrote the paper: EVF CJS JRR.
The epidermal growth factor receptor (EGFR)-targeting IgG1 monoclonal antibody, cetuximab, is a breakthrough in targeted therapy for head and neck cancers, especially among patients with recurrent or metastatic disease [1]. In patients with locally advanced head and neck cancer, radiotherapy in combination with cetuximab has prolonged the median overall survival in a statistically significant manner when compared to radiotherapy alone [2]. In head and neck cancer patients with recurrent or metastatic squamous cell carcinoma, cetuximab in combination with platinum-fluorouracil chemotherapy improved overall survival when given as first-line treatment [3]. Recently, cisplatin-based chemoradiation in combination with cetuximab led to a complete response rate of 71 among participants in a phase II study that enrolled advanced head and neck cancer patients [4].Previous studies reported that the administration of cetuximab does not al.Ure S3 Western blot of PK-resistant fragments. In unpurified (1) and purified GPI- PrPSc (2). Both samples were digested with proteinase K, 25 mg/ml and 10 mg/ml, respectively, treated with PNGase F and resolved on a Tricine-SDS-PAGE gel. WB was probed with the R1 antibody. (TIF) Figure S4 Bayesian protein reconstruction of the nanoLC-ESI-MS spectra of PK-treated purified GPI2 PrPSc. The mass graphs of the three peaks: 17148 Da (top), 16729 Da (middle) and 16371 Da (bottom), identified by ESI-TOF are shown. (TIF) Figure S5 Western blot of recombinant MoPrP(23?31)cleavage by PK. Samples were digested with different concentrations of PK: 0, 0.2, 1, 5, 10 and 25 mg/ml. Samples were subjected to Tricine-SDS-PAGE and the blot was probed with R1 antibody. (TIF)Figure S6 Schematic representations of the data. A. A scheme of GPI2 PrP sequence, showing the PK-resistant areas (blue squares) and the PK cleavage points and flexible areas (gray line). B. Lengthwise comparison of the different peptides found by limited proteolysis and MALDI-TOF analysis (colors match those displayed in Figure 2). (TIF)Acknowledgments ImmunohistochemistryImmediately after extraction, the brain was fixed in formalin and then sliced into four transversal sections by cutting the brain caudally and rostrally to the midbrain and at the level of the basal nuclei. The sections were dehydrated by equilibration in solutions of progressively higher ethanol concentration and then equilibrated with xylene before being embedded in paraffin. Haematoxylineosin was used to stain the 4 mm thick sections. Additional sections were mounted on 3-triethoxysilyl-propylamine-coated glass slides for immunohistochemical (IHC) studies. These brain sections were deparaffinised, immersed in formic acid containing peroxidase inhibitors, and autoclaved prior to IHC analysis. These autoclaved samples were washed, treatedWe thank Bruce Chesebro, Rocky Mountain Laboratory, NIH, MT, USA, for his kind gift of GPI- mice, Hanna Serban, Institute for Neurodegenerative Diseases, UCSF, CA, USA, for generously providing antibody R1, ?Juan Maria Torres, CISA, Madrid, Spain, for RML inoculum, Valerie Sim, University of Alberta, Edmonton, Canada, for advice on GPI- PrPSc isolation and Melissa L. Erickson, USDA, for help in preparing the manuscript.Author ContributionsConceived and designed the experiments: EVF JA CJS JRR. Performed the experiments: EVF JA EV ID. Analyzed the data: EVF JA EV CJS JRR. Contributed reagents/materials/analysis tools: MAP AR LS BP. Wrote the paper: EVF CJS JRR.
The epidermal growth factor receptor (EGFR)-targeting IgG1 monoclonal antibody, cetuximab, is a breakthrough in targeted therapy for head and neck cancers, especially among patients with recurrent or metastatic disease [1]. In patients with locally advanced head and neck cancer, radiotherapy in combination with cetuximab has prolonged the median overall survival in a statistically significant manner when compared to radiotherapy alone [2]. In head and neck cancer patients with recurrent or metastatic squamous cell carcinoma, cetuximab in combination with platinum-fluorouracil chemotherapy improved overall survival when given as first-line treatment [3]. Recently, cisplatin-based chemoradiation in combination with cetuximab led to a complete response rate of 71 among participants in a phase II study that enrolled advanced head and neck cancer patients [4].Previous studies reported that the administration of cetuximab does not al.