Journal: Protein science : a publication of the Protein Society

Article Title: Structural features of thyroglobulin linked to protein trafficking.

doi: 10.1002/pro.4784

Figure Lengend Snippet: FIGURE 2 bTg protein export upon disruption of the C408–C608 disulfide bridge. (a) Schematic of bTg wild-type (WT) and mutants studied in this figure. (b) 293 T were either nontransfected (NT) or transfected to express the indicated constructs. After a 13-h period, the media (M) were collected and cells (C) were lysed and analyzed by SDS-PAGE under nonreducing (left) and reducing (right) conditions, electrotransferred to nitrocellulose, and immunoblotted with polyclonal anti-Tg. HSP90 is a loading control. (c) From three independent experiments, recovery of Tg in 293 T cell lysates (dark gray) and media (light gray) were quantified for each construct from reducing SDS- PAGE analysis; mean ± SD. No significant differences were observed between groups.

Article Snippet: Rabbit polyclonal anti-Tg was as described (Kim & Arvan, 1991), rabbit polyclonal anti-C-Myc (RMYC-45A) from ICL, mouse monoclonal anti-beta-actin (sc-47778) and anti-HSP90α/β (sc-13119) from Santa Cruz Biotechnology.

Techniques: Disruption, Transfection, Construct, SDS Page, Control

Journal: Protein science : a publication of the Protein Society

Article Title: Structural features of thyroglobulin linked to protein trafficking.

doi: 10.1002/pro.4784

Figure Lengend Snippet: FIGURE 4 Functional role of K2225 in secretory ChEL to rescue the secretory trafficking of co-expressed Tg-I–II–III (that lacks its own ChEL). (a) Schematic representation of wild-type (WT) and mutant secretory ChEL: the white box represents an engineered signal peptide to make ChEL into a secretory protein. (b) 293 T were either nontransfected (NT) or transfected to express the indicated mouse Tg-derived constructs (Tables S1 and S2). After a 13-h period, the media (M) were collected and cells (C) were lysed and analyzed by SDS-PAGE and Western blotting with polyclonal anti-Tg. β-actin is a loading control. (c) From three independent experiments like that shown in panel (b), the relative secretion of ChEL was calculated for each construct; mean ± SD; N.S = non-significant. (d) 293 T were nontransfected (NT), transfected with Tg region I–II–III alone or co-expressed with the indicated secretory ChEL construct. C = cells; M = media. β-actin is a loading control. (e, f). From three independent experiments like that shown in panel (d), the relative secretion of ChEL (panel e) and I–II– III (panel f) was calculated; mean ± SD; N.S = non-significant; *, p < 0.05.

Article Snippet: Rabbit polyclonal anti-Tg was as described (Kim & Arvan, 1991), rabbit polyclonal anti-C-Myc (RMYC-45A) from ICL, mouse monoclonal anti-beta-actin (sc-47778) and anti-HSP90α/β (sc-13119) from Santa Cruz Biotechnology.

Techniques: Functional Assay, Mutagenesis, Transfection, Derivative Assay, Construct, SDS Page, Western Blot, Control

Journal: Protein science : a publication of the Protein Society

Article Title: Structural features of thyroglobulin linked to protein trafficking.

doi: 10.1002/pro.4784

Figure Lengend Snippet: FIGURE 5 Functional role of K2225 in the trafficking of full-length Tg. (a) Schematic representation of wild-type (WT) Tg and Tg- K2225E (for amino acid numbering differences between species, see Table S2). (b) 293 T were nontransfected (NT) or transfected to express the indicated mouse Tg constructs. After a 13-h period, the media (M) were collected and cells (C) were lysed and analyzed by SDS-PAGE under nonreducing (left) and reducing (right) conditions, electrotransferred to nitrocellulose, and immunoblotted with polyclonal anti-Tg. HSP90 is a loading control. (c) From three independent experiments like that shown in panel (b) (reduced samples), the relative secretion of mouse Tg was calculated for each construct; mean ± SD; *, p < 0.05.

Article Snippet: Rabbit polyclonal anti-Tg was as described (Kim & Arvan, 1991), rabbit polyclonal anti-C-Myc (RMYC-45A) from ICL, mouse monoclonal anti-beta-actin (sc-47778) and anti-HSP90α/β (sc-13119) from Santa Cruz Biotechnology.

Techniques: Functional Assay, Transfection, Construct, SDS Page, Control

Journal: Protein science : a publication of the Protein Society

Article Title: Structural features of thyroglobulin linked to protein trafficking.

doi: 10.1002/pro.4784

Figure Lengend Snippet: FIGURE 6 Role of the C2444–C2455 disulfide bond in the trafficking of secretory ChEL. (a) Schematic representation of wild-type (WT) or mutant secretory ChEL constructs. The putative C2444–C2455 disulfide loop in the ChEL domain is circled above. For amino acid numbering differences between species, see Table S2. (b) 293 T were either nontransfected (NT) or transfected to express the indicated secretory ChEL constructs derived from mouse Tg. After a 13-h period, the media (M) were collected and cell (C) were lysed and analyzed by SDS-PAGE under nonreducing (left) and reducing (right) conditions, electrotransferred to nitrocellulose, and immunoblotted with polyclonal anti-Tg. β-actin is a loading control. (c) From three independent experiments like that shown in panel (b) (reduced samples), the relative secretion of secretory ChEL was calculated for each construct; mean ± SD; *, p < 0.05.

Article Snippet: Rabbit polyclonal anti-Tg was as described (Kim & Arvan, 1991), rabbit polyclonal anti-C-Myc (RMYC-45A) from ICL, mouse monoclonal anti-beta-actin (sc-47778) and anti-HSP90α/β (sc-13119) from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Construct, Transfection, Derivative Assay, SDS Page, Control

Journal: Protein science : a publication of the Protein Society

Article Title: Structural features of thyroglobulin linked to protein trafficking.

doi: 10.1002/pro.4784

Figure Lengend Snippet: FIGURE 7 Role of the C2444–C2455 disulfide bond in the trafficking of full-length Tg. (a) Schematic representation of wild-type (WT) or mutant Tg constructs. The putative C2444–C2455 disulfide loop in the Tg ChEL domain is circled above. For amino acid numbering differences between species, see Table S2. (b) 293 T were either nontransfected (NT) or transfected to express the indicated bTg constructs. After a 13-h period, the media (M) were collected and cells (C) were lysed and analyzed by SDS-PAGE under nonreducing (left) and reducing (right) conditions, electrotransferred to nitrocellulose, and immunoblotted with polyclonal anti-Tg. HSP90 is a loading control. (c) From three independent experiments like that shown in panel (b) (reduced samples), the relative secretion of bTg was calculated for each construct; mean ± SD; *, p < 0.05.

Article Snippet: Rabbit polyclonal anti-Tg was as described (Kim & Arvan, 1991), rabbit polyclonal anti-C-Myc (RMYC-45A) from ICL, mouse monoclonal anti-beta-actin (sc-47778) and anti-HSP90α/β (sc-13119) from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Construct, Transfection, SDS Page, Control

Journal: Protein science : a publication of the Protein Society

Article Title: Structural features of thyroglobulin linked to protein trafficking.

doi: 10.1002/pro.4784

Figure Lengend Snippet: FIGURE 8 Absence of the C2444–C2455 disulfide bond affects folding of the ChEL domain. (a) 293 T were either nontransfected (NT) or transfected to express the indicated secretory ChEL constructs derived from mouse Tg. (For amino acid numbering differences between species, see Table S2.) After a 13 h period, the media (M) were collected and cells (C) were lysed and analyzed by SDS-PAGE under nonreducing (left) and reducing (right) conditions, electrotransferred to nitrocellulose, and immunoblotted with polyclonal anti-Tg. β-actin is a loading control. Bands detected from NT cells (and media) are nonspecific. By nonreducing SDS-PAGE, intracellular accumulation of misfolded mutant ChEL monomers and aberrant disulfide-linked complexes were detected. (b) Lysates from transfected 293 T cells expressing the indicated constructs were analyzed by SDS-PAGE under nonreducing (left) and reducing (right) conditions, followed by Western blotting with polyclonal anti-Tg. β-actin is a loading control. Notably, the ChEL-K225E construct does not form the aberrant disulfide-linked complexes that are detected for the other two misfolded mutant ChEL constructs.

Article Snippet: Rabbit polyclonal anti-Tg was as described (Kim & Arvan, 1991), rabbit polyclonal anti-C-Myc (RMYC-45A) from ICL, mouse monoclonal anti-beta-actin (sc-47778) and anti-HSP90α/β (sc-13119) from Santa Cruz Biotechnology.

Techniques: Transfection, Construct, Derivative Assay, SDS Page, Control, Mutagenesis, Expressing, Western Blot

Journal: Oncogene

Article Title: Glioma-specific Domain IV EGFR cysteine mutations promote ligand-induced covalent receptor dimerization and display enhanced sensitivity to dacomitinib in vivo.

doi: 10.1038/onc.2014.106

Figure Lengend Snippet: Figure 1. Biochemical characterization of Domain IV mutants. (a) Representative flow cytometry histograms for cell-surface EGFR expression, as determined by mAb528 binding after viral transduction of U87MG cells with vectors expressing the indicated mutants and subsequent drug selection. No cell sorting was conducted on the Domain IV mutant cell lines. (b) Eu-pY assay for sensitive detection of basal phosphorylation. Each cell line was serum-starved in the presence of vehicle, 1 μM lapatinib or 40 μg/ml EGFR 501-Fc ligand trap overnight before the assay was conducted. Data is presented as the mean time-resolved fluorescence (TRF) obtained for each test group over quadruple-technical replicates ± standard error (s.e.). Experiments were repeated three times. (c) Eu-pY assay for determination of global phosphotyrosine status of receptor in response to titration of ligand. Each cell line was serum-starved and treated with titrated concentrations of EGF (first graph), TGF-α (second graph), heparin-bound EGF (third graph) or betacellulin (fourth graph). Basal phosphorylation TRF reads were subtracted from all readings and then readings graphed as a percentage TRF obtained for each ligand concentration compared with the maximum stimulus obtained at 10 or 30 nM± s.e. Experiments were repeated three times.

Article Snippet: The anti-C-terminal EGFR (clone 1005) rabbit polyclonal antibody, either alone or conjugated to agarose beads, was from Santa Cruz (Dallas, TX, USA).

Techniques: Cytometry, Expressing, Binding Assay, Transduction, Selection, FACS, Mutagenesis, Phospho-proteomics, Titration, Concentration Assay

Journal: Oncogene

Article Title: Glioma-specific Domain IV EGFR cysteine mutations promote ligand-induced covalent receptor dimerization and display enhanced sensitivity to dacomitinib in vivo.

doi: 10.1038/onc.2014.106

Figure Lengend Snippet: Figure 2. Ligand-induced covalent dimerization of Domain IV EGFR mutants. (a–c) Nonreducing and reducing western blot analyzes for total EGFR (top panels) and pY1173 EGFR (middle panels). Overlays of the EGFR (red) and pY1173 (green) signal are shown to determine the active species (bottom panels). Dimeric (D) and monomeric (M) species for both wtEGFR, mutant EGFR and EGFRvIII are depicted by arrows. (a) The control cell line (U87MG) and cell lines expressing wtEGFR and EGFRvIII, treated with vehicle or EGF. (b) Cell lines expressing the Domain IV mutants C620Y, C624F, C628Y and C636Y, treated with vehicle or EGF. (c) Nonreducing western blot analyzes for total EGFR (top panels) and pY1173 EGFR (middle panels) in cell lines expressing wtEGFR, C620Y and C624F treated with the indicated ligands. In all figures, a pan-actin loading control is included.

Article Snippet: The anti-C-terminal EGFR (clone 1005) rabbit polyclonal antibody, either alone or conjugated to agarose beads, was from Santa Cruz (Dallas, TX, USA).

Techniques: Western Blot, Mutagenesis, Control, Expressing

Journal: Oncogene

Article Title: Glioma-specific Domain IV EGFR cysteine mutations promote ligand-induced covalent receptor dimerization and display enhanced sensitivity to dacomitinib in vivo.

doi: 10.1038/onc.2014.106

Figure Lengend Snippet: Figure 3. Analysis of cell-surface species present on Domain IV mutant cell lines. (a) Cells expressing wtEGFR, C620Y or C624F were treated at 4 °C with vehicle or EGF and the cell surface subsequently biotinylated. Streptavidin immunoprecipitations were performed after which western blot analyzes were performed for total EGFR (top panel) or pY1173 EGFR (middle panel), with overlays of the total EGFR (red) and pY1173 (green) signal shown to determine the active species (bottom panel). Dimeric (D) and monomeric (M) species are depicted by arrows. (b) Reciprocal experiments involving cell- surface biotinylation followed by EGFR immunoprecipitation and then western blot analyzes for streptavidin binding, indicating the total EGFR cell-surface species present. Dimeric (D) and monomeric (M) species are depicted by arrows. (c) wtEGFR, C620Y and C624F cell lines were cultured with or without 2 μg/ml of swainsonine overnight before cell lysis and western blot analyzes for total EGFR using an ECD- (top panel) or C-terminal domain-specific antibodies (middle panel). Dimeric (D) and monomeric (M) species are depicted by arrows. A pan-actin control was included for loading (bottom panel).

Article Snippet: The anti-C-terminal EGFR (clone 1005) rabbit polyclonal antibody, either alone or conjugated to agarose beads, was from Santa Cruz (Dallas, TX, USA).

Techniques: Mutagenesis, Expressing, Western Blot, Immunoprecipitation, Binding Assay, Cell Culture, Lysis, Control

Journal: Oncogene

Article Title: Glioma-specific Domain IV EGFR cysteine mutations promote ligand-induced covalent receptor dimerization and display enhanced sensitivity to dacomitinib in vivo.

doi: 10.1038/onc.2014.106

Figure Lengend Snippet: Figure 4. Dacomitinib-induced cytoplasmic retention of receptor and loss of ligand binding. (a) Cells expressing wtEGFR, C620Y, C624F or EGFRvIII were treated overnight with vehicle or 1 μM of gefitinib, dacomitinib or lapatinib. The EGF-alone control tests were stimulated the next day and cell lysates probed by nonreducing western blot analyzes for total EGFR (top panels) or pY1173 EGFR (middle panels). An overlay of the total EGFR (red) and pY1173 (green) signal are shown to determine the active species (bottom panels). Dimeric (D) and monomeric (M) species are depicted by arrows. (b) Representative images for cell-surface EGFR immunofluorescence conducted following overnight treatment of cells expressing wtEGFR, C620Y, EGFRvIII or C16S with vehicle (top panels) or 1 μM dacomitinib (bottom panels). Blue, nucleus; green, EGFR. Scale bar, 20 μm. (c) Cell-surface flow cytometry for EGFR status, as determined by panitumumab binding. Cells were treated overnight with vehicle or 1 μM dacomitinib, harvested and fixed before staining with isotype-control antibody or panitumumab. Red, isotype staining; blue, panitumumab staining in vehicle-treated cells; green, panitumumab staining in dacomitinib-treated cells. (d) Eu-EGF binding to cells after overnight treatment with vehicle or 1 μM dacomitinib. TRF readings were obtained for quadruple-technical replicates and graphed as a mean percentage of the vehicle in each cell line ± s.e. * represents significant differences compared with the respective vehicle control. Experiments were repeated three times.

Article Snippet: The anti-C-terminal EGFR (clone 1005) rabbit polyclonal antibody, either alone or conjugated to agarose beads, was from Santa Cruz (Dallas, TX, USA).

Techniques: Ligand Binding Assay, Expressing, Control, Western Blot, Cytometry, Binding Assay, Staining