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primary antibodies against total egfr  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc primary antibodies against total egfr
    Effects of Ncs on apoptosis and cell cycle. ( A ) Cells were treated with 50 nM Ncs for 24 h and stained with calcein-AM/PI, followed by image capture using fluorescence microscopy (green: live cells, red; dead cells), scale bars = 50 μm. ( B ) cells were treated with 50 nM Ncs for 24 h and stained with annexin V and PI and followed by flow cytometric analysis of apoptosis. Apoptotic cells were quantified with early and late apoptotic cell populations. ( C ) For cell cycle arrays, 50 nM Ncs-treated cells were fixed with 70% ethanol and stained with PI, followed by flow cytometry analysis. ( D and E ) Cells were treated with indicated concentrations (0–100 nM) of Ncs for 24 h and cell lysates were subjected to immunoblotting analysis using the <t>indicated</t> <t>antibodies.</t> Expression levels of <t>p-EGFR</t> and p-ERK1/2 were quantified from immunoblot bands and normalized to total EGFR and total ERK1/2 expression levels, respectively ( D ). Data were presented as mean values from three independent experiments and error bars represent standard deviations. * p < 0.05, ** p < 0.01
    Primary Antibodies Against Total Egfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1608 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/primary+antibodies+against+total+egfr/EGF+Receptor+XP+Rabbit+mAb/pmc12679757-55-0-6
    Average 96 stars, based on 1608 article reviews
    primary antibodies against total egfr - by Bioz Stars, 2026-09
    96/100 stars

    Images

    1) Product Images from "Narciclasine as a potential therapeutic agent to overcome EGFR-TKI resistance in non-small cell lung cancer"

    Article Title: Narciclasine as a potential therapeutic agent to overcome EGFR-TKI resistance in non-small cell lung cancer

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-025-07368-4

    Effects of Ncs on apoptosis and cell cycle. ( A ) Cells were treated with 50 nM Ncs for 24 h and stained with calcein-AM/PI, followed by image capture using fluorescence microscopy (green: live cells, red; dead cells), scale bars = 50 μm. ( B ) cells were treated with 50 nM Ncs for 24 h and stained with annexin V and PI and followed by flow cytometric analysis of apoptosis. Apoptotic cells were quantified with early and late apoptotic cell populations. ( C ) For cell cycle arrays, 50 nM Ncs-treated cells were fixed with 70% ethanol and stained with PI, followed by flow cytometry analysis. ( D and E ) Cells were treated with indicated concentrations (0–100 nM) of Ncs for 24 h and cell lysates were subjected to immunoblotting analysis using the indicated antibodies. Expression levels of p-EGFR and p-ERK1/2 were quantified from immunoblot bands and normalized to total EGFR and total ERK1/2 expression levels, respectively ( D ). Data were presented as mean values from three independent experiments and error bars represent standard deviations. * p < 0.05, ** p < 0.01
    Figure Legend Snippet: Effects of Ncs on apoptosis and cell cycle. ( A ) Cells were treated with 50 nM Ncs for 24 h and stained with calcein-AM/PI, followed by image capture using fluorescence microscopy (green: live cells, red; dead cells), scale bars = 50 μm. ( B ) cells were treated with 50 nM Ncs for 24 h and stained with annexin V and PI and followed by flow cytometric analysis of apoptosis. Apoptotic cells were quantified with early and late apoptotic cell populations. ( C ) For cell cycle arrays, 50 nM Ncs-treated cells were fixed with 70% ethanol and stained with PI, followed by flow cytometry analysis. ( D and E ) Cells were treated with indicated concentrations (0–100 nM) of Ncs for 24 h and cell lysates were subjected to immunoblotting analysis using the indicated antibodies. Expression levels of p-EGFR and p-ERK1/2 were quantified from immunoblot bands and normalized to total EGFR and total ERK1/2 expression levels, respectively ( D ). Data were presented as mean values from three independent experiments and error bars represent standard deviations. * p < 0.05, ** p < 0.01

    Techniques Used: Staining, Fluorescence, Microscopy, Flow Cytometry, Western Blot, Expressing

    Effects of Ncs on cell growth in PC-9 and PC-9-GR cells. ( A ) PC-9 and PC-9-GR cells (3 × 10 3 cells/well) were plated in 96-well plates and treated with gefitinib in a dose-dependent manner for 48 h. ( B ) Cells were treated with Ncs at the indicated concentrations for 48 hours, and cell growth rates were measured using the WST-1 assay. ( C ) Cells were treated with Ncs for 48 hours, and representative images are shown. ( D ) Cells (1 × 10 4 cells/well) were plated in 24-well plates and treated with Ncs at the indicated concentrations for 48 h. Live and dead cells were counted using the trypan blue staining assay. ( E and F ) Cells were treated with Ncs for 48 h, fixed with 70% ethanol, stained with PI, and analyzed by flow cytometry to assess cell cycle distribution. ( G ) PC-9 and PC-9-GR cells were treated with the indicated concentrations (0-50 nM) of Ncs for 24 h. Cell lysates were then subjected to immunoblotting analysis using the indicated antibodies. Expression levels of p-EGFR, p-Src, and p-STAT3 were quantified and normalized to total EGFR, total Src, and total STAT3 expression levels, respectively. Data were presented as mean values from three independent experiments and error bars represent standard deviations of the mean of three measurements. Magnification: x 100, n.s. >0.05, *P < 0.05, **P < 0.01
    Figure Legend Snippet: Effects of Ncs on cell growth in PC-9 and PC-9-GR cells. ( A ) PC-9 and PC-9-GR cells (3 × 10 3 cells/well) were plated in 96-well plates and treated with gefitinib in a dose-dependent manner for 48 h. ( B ) Cells were treated with Ncs at the indicated concentrations for 48 hours, and cell growth rates were measured using the WST-1 assay. ( C ) Cells were treated with Ncs for 48 hours, and representative images are shown. ( D ) Cells (1 × 10 4 cells/well) were plated in 24-well plates and treated with Ncs at the indicated concentrations for 48 h. Live and dead cells were counted using the trypan blue staining assay. ( E and F ) Cells were treated with Ncs for 48 h, fixed with 70% ethanol, stained with PI, and analyzed by flow cytometry to assess cell cycle distribution. ( G ) PC-9 and PC-9-GR cells were treated with the indicated concentrations (0-50 nM) of Ncs for 24 h. Cell lysates were then subjected to immunoblotting analysis using the indicated antibodies. Expression levels of p-EGFR, p-Src, and p-STAT3 were quantified and normalized to total EGFR, total Src, and total STAT3 expression levels, respectively. Data were presented as mean values from three independent experiments and error bars represent standard deviations of the mean of three measurements. Magnification: x 100, n.s. >0.05, *P < 0.05, **P < 0.01

    Techniques Used: WST-1 Assay, Staining, Flow Cytometry, Western Blot, Expressing

    Effects of Ncs on EGFR activity and EGFR signaling. ( A ) H1975 cells were treated with 50 nM of Ncs for indicated times and cell lysates were subjected to immunoblotting analysis using the indicated antibodies. ( B ) Cells were treated with 50 nM of Ncs for the indicated durations, and total RNA was isolated. EGFR mRNA levels were determined by real-time PCR analysis and normalized to GAPDH expression. ( C ) Cells were treated with 20 μM of cycloheximide (CHX) for the indicated time points in the presence of either DMSO or 50 nM of Ncs for 24 h. Cell lysates were subjected to immunoblotting analysis using the indicated antibodies (upper panel). EGFR expression levels were quantified from immunoblot bands and normalized to GAPDH, comparing CHX treatment in the presence of DMSO or Ncs (lower panel). ( D ) Cells were treated with 50 nM of Ncs for 24 h in the presence of BafA1 (20 nM, 12 h) or MG132 (20 μM, 2 h). Cell lysates were subjected to immunoblotting analysis using indicated antibodies. ( E ) Cells were plated on coverslips and treated with DMSO (control) or 50 nM of Ncs for 24 h. Cells were then fixed with 4% paraformaldehyde (PFA) and subjected to immunofluorescence staining using DAPI, EGFR, and LAMP1 antibody. ( F ) Cells were treated with 50 nM of Ncs for 24 h, followed by EGF (10 nM, 15 min) stimulation. Cell lysates were subjected to immunoblotting analysis using the specified antibodies. Similar results were observed in three independent experiments. Error bars represent standard deviations of the mean of three measurements, *P < 0.05, **P < 0.01, ***P < 0.001
    Figure Legend Snippet: Effects of Ncs on EGFR activity and EGFR signaling. ( A ) H1975 cells were treated with 50 nM of Ncs for indicated times and cell lysates were subjected to immunoblotting analysis using the indicated antibodies. ( B ) Cells were treated with 50 nM of Ncs for the indicated durations, and total RNA was isolated. EGFR mRNA levels were determined by real-time PCR analysis and normalized to GAPDH expression. ( C ) Cells were treated with 20 μM of cycloheximide (CHX) for the indicated time points in the presence of either DMSO or 50 nM of Ncs for 24 h. Cell lysates were subjected to immunoblotting analysis using the indicated antibodies (upper panel). EGFR expression levels were quantified from immunoblot bands and normalized to GAPDH, comparing CHX treatment in the presence of DMSO or Ncs (lower panel). ( D ) Cells were treated with 50 nM of Ncs for 24 h in the presence of BafA1 (20 nM, 12 h) or MG132 (20 μM, 2 h). Cell lysates were subjected to immunoblotting analysis using indicated antibodies. ( E ) Cells were plated on coverslips and treated with DMSO (control) or 50 nM of Ncs for 24 h. Cells were then fixed with 4% paraformaldehyde (PFA) and subjected to immunofluorescence staining using DAPI, EGFR, and LAMP1 antibody. ( F ) Cells were treated with 50 nM of Ncs for 24 h, followed by EGF (10 nM, 15 min) stimulation. Cell lysates were subjected to immunoblotting analysis using the specified antibodies. Similar results were observed in three independent experiments. Error bars represent standard deviations of the mean of three measurements, *P < 0.05, **P < 0.01, ***P < 0.001

    Techniques Used: Activity Assay, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Control, Immunofluorescence, Staining

    Anti-cancer effects of ncs in vivo. ( A ) Adult-stage C. elegans models: wild type (WT) and jgIs25 strains. The jgIs25 strain exhibits polyps on the ventral side (arrows in the lower panel). Scale bar = 20 μm. ( B ) Effects of gefitinib and ncs treatment on jgIs25 . the arrows in the upper panel indicate polyps, and the rectangular area is magnified in the lower panel. Scale bar = 200 μm (upper), 20 μm (lower). ( C ) Quantification of polyp formation in jgIs25 following treatment with the indicated drugs. ( D and G ) In vivo tumor model using subcutaneous transplantation of A549 and H1975 cells into nude mice, followed by ncs treatment. Tumor volumes were measured at the indicated time points in control and ncs-treated mice. Tumor weights were measured after sacrifice. ( E and H ) Representative image of xenograft tumors after sacrifice. ( F and I ) Immunohistochemical (IHC) staining of xenograft tumors using anti-EGFR, anti-phospho-EGFR (Y1068), and anti-phospho-STAT3 (Y705) antibodies. Scale bar = 500 μm. Similar results were observed in three independent experiments. Error bars represent standard deviations of the mean of three measurements, *p < 0.05, **p < 0.01, *p < 0.001
    Figure Legend Snippet: Anti-cancer effects of ncs in vivo. ( A ) Adult-stage C. elegans models: wild type (WT) and jgIs25 strains. The jgIs25 strain exhibits polyps on the ventral side (arrows in the lower panel). Scale bar = 20 μm. ( B ) Effects of gefitinib and ncs treatment on jgIs25 . the arrows in the upper panel indicate polyps, and the rectangular area is magnified in the lower panel. Scale bar = 200 μm (upper), 20 μm (lower). ( C ) Quantification of polyp formation in jgIs25 following treatment with the indicated drugs. ( D and G ) In vivo tumor model using subcutaneous transplantation of A549 and H1975 cells into nude mice, followed by ncs treatment. Tumor volumes were measured at the indicated time points in control and ncs-treated mice. Tumor weights were measured after sacrifice. ( E and H ) Representative image of xenograft tumors after sacrifice. ( F and I ) Immunohistochemical (IHC) staining of xenograft tumors using anti-EGFR, anti-phospho-EGFR (Y1068), and anti-phospho-STAT3 (Y705) antibodies. Scale bar = 500 μm. Similar results were observed in three independent experiments. Error bars represent standard deviations of the mean of three measurements, *p < 0.05, **p < 0.01, *p < 0.001

    Techniques Used: In Vivo, Transplantation Assay, Control, Immunohistochemical staining, Immunohistochemistry

    Related Articles

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    Article Title: PPARα-mediated lipid metabolism reprogramming supports anti-EGFR therapy resistance in head and neck squamous cell carcinoma
    Article Snippet: Novel plant genotypes Seed stocks Authentication Plants For Western blot analyses, primary antibodies against !-actin (clone AC-15, #A5441, Sigma-Aldrich; 1:10,000), phosphorylated (Ser-79; clone D7D11, #11818, Cell Signaling Technology; 1:1,000) and total acetyl-CoA carboxylase (clone C83B10, #3676, Cell Signaling Technology; 1:1,000), CD36 (#NB400-144, Novus Biologicals; 1:1,000), CPT1A (clone D3B3, #12252, Cell Signaling Technology; 1:1,000), phosphorylated (Tyr-1068; clone D7A5, #3777, Cell Signaling Technology; 1:1,000) and total EGFR (clone D38B1, #4267, Cell Signaling Technology; 1:1,000), fatty acid synthase (clone C20G5, #3180, Cell Signaling Technology; 1:1,000), HSP90 (clone 68, #610419, BD Biosciences; 1:7,500) and SCD1 (clone CD.E10, #ab19862, Abcam, 1:1,000). were all incubated in 5% BSA overnight at 4°C.

    Western Blot:

    Article Title: Narciclasine as a potential therapeutic agent to overcome EGFR-TKI resistance in non-small cell lung cancer.
    Article Snippet: .. Primary antibodies against total EGFR (#4267, Cell Signaling Technology(CST)), CDK1 (#9116, CST), phospho-EGFR Y1068 (#44-788 G, Invitrogen), total STAT3 (#4904, CST), phospho-STAT3 Y705 (#9145, CST), phospho-Src (#6943,CST), total-Src (#2109, CST), phospho-CDK1 Y15 (#4539, CST), phospho-Wee1 (#4910, CST), Myt1 (#4282, CST), p21 (#2947, CST), Cdc25B (#9525, CST), GAPDH (#5174, CST), β-actin (#A700057, Bethyl Laboratory), phospho-ERK1/2 (#9101, CST), Bcl-2 (#sc-7382, Santa Cruz), and c-Myc (#sc-789, Santa Cruz) were used for immunoblotting. .. Horseradish peroxidase (HRP)-conjugated rabbit IgG and HRP-conjugated mouse IgG were purchased from Enzo Life Sciences (Farmingdale, NY).

    Article Title: Narciclasine as a potential therapeutic agent to overcome EGFR-TKI resistance in non-small cell lung cancer
    Article Snippet: .. Primary antibodies against total EGFR (#4267, Cell Signaling Technology(CST)), CDK1 (#9116, CST), phospho-EGFR Y1068 (#44-788 G, Invitrogen), total STAT3 (#4904, CST), phospho-STAT3 Y705 (#9145, CST), phospho-Src (#6943,CST), total-Src (#2109, CST), phospho-CDK1 Y15 (#4539, CST), phospho-Wee1 (#4910, CST), Myt1 (#4282, CST), p21 (#2947, CST), Cdc25B (#9525, CST), GAPDH (#5174, CST), β-actin (#A700-057, Bethyl Laboratory), phospho-ERK1/2 (#9101, CST), Bcl-2 (#sc-7382, Santa Cruz), and c-Myc (#sc-789, Santa Cruz) were used for immunoblotting. .. Horseradish peroxidase (HRP)-conjugated rabbit IgG and HRP-conjugated mouse IgG were purchased from Enzo Life Sciences (Farmingdale, NY).



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    Effects of Ncs on apoptosis and cell cycle. ( A ) Cells were treated with 50 nM Ncs for 24 h and stained with calcein-AM/PI, followed by image capture using fluorescence microscopy (green: live cells, red; dead cells), scale bars = 50 μm. ( B ) cells were treated with 50 nM Ncs for 24 h and stained with annexin V and PI and followed by flow cytometric analysis of apoptosis. Apoptotic cells were quantified with early and late apoptotic cell populations. ( C ) For cell cycle arrays, 50 nM Ncs-treated cells were fixed with 70% ethanol and stained with PI, followed by flow cytometry analysis. ( D and E ) Cells were treated with indicated concentrations (0–100 nM) of Ncs for 24 h and cell lysates were subjected to immunoblotting analysis using the indicated antibodies. Expression levels of p-EGFR and p-ERK1/2 were quantified from immunoblot bands and normalized to total EGFR and total ERK1/2 expression levels, respectively ( D ). Data were presented as mean values from three independent experiments and error bars represent standard deviations. * p < 0.05, ** p < 0.01

    Journal: Journal of Translational Medicine

    Article Title: Narciclasine as a potential therapeutic agent to overcome EGFR-TKI resistance in non-small cell lung cancer

    doi: 10.1186/s12967-025-07368-4

    Figure Lengend Snippet: Effects of Ncs on apoptosis and cell cycle. ( A ) Cells were treated with 50 nM Ncs for 24 h and stained with calcein-AM/PI, followed by image capture using fluorescence microscopy (green: live cells, red; dead cells), scale bars = 50 μm. ( B ) cells were treated with 50 nM Ncs for 24 h and stained with annexin V and PI and followed by flow cytometric analysis of apoptosis. Apoptotic cells were quantified with early and late apoptotic cell populations. ( C ) For cell cycle arrays, 50 nM Ncs-treated cells were fixed with 70% ethanol and stained with PI, followed by flow cytometry analysis. ( D and E ) Cells were treated with indicated concentrations (0–100 nM) of Ncs for 24 h and cell lysates were subjected to immunoblotting analysis using the indicated antibodies. Expression levels of p-EGFR and p-ERK1/2 were quantified from immunoblot bands and normalized to total EGFR and total ERK1/2 expression levels, respectively ( D ). Data were presented as mean values from three independent experiments and error bars represent standard deviations. * p < 0.05, ** p < 0.01

    Article Snippet: Primary antibodies against total EGFR (#4267, Cell Signaling Technology(CST)), CDK1 (#9116, CST), phospho-EGFR Y1068 (#44-788 G, Invitrogen), total STAT3 (#4904, CST), phospho-STAT3 Y705 (#9145, CST), phospho-Src (#6943,CST), total-Src (#2109, CST), phospho-CDK1 Y15 (#4539, CST), phospho-Wee1 (#4910, CST), Myt1 (#4282, CST), p21 (#2947, CST), Cdc25B (#9525, CST), GAPDH (#5174, CST), β-actin (#A700-057, Bethyl Laboratory), phospho-ERK1/2 (#9101, CST), Bcl-2 (#sc-7382, Santa Cruz), and c-Myc (#sc-789, Santa Cruz) were used for immunoblotting.

    Techniques: Staining, Fluorescence, Microscopy, Flow Cytometry, Western Blot, Expressing

    Effects of Ncs on cell growth in PC-9 and PC-9-GR cells. ( A ) PC-9 and PC-9-GR cells (3 × 10 3 cells/well) were plated in 96-well plates and treated with gefitinib in a dose-dependent manner for 48 h. ( B ) Cells were treated with Ncs at the indicated concentrations for 48 hours, and cell growth rates were measured using the WST-1 assay. ( C ) Cells were treated with Ncs for 48 hours, and representative images are shown. ( D ) Cells (1 × 10 4 cells/well) were plated in 24-well plates and treated with Ncs at the indicated concentrations for 48 h. Live and dead cells were counted using the trypan blue staining assay. ( E and F ) Cells were treated with Ncs for 48 h, fixed with 70% ethanol, stained with PI, and analyzed by flow cytometry to assess cell cycle distribution. ( G ) PC-9 and PC-9-GR cells were treated with the indicated concentrations (0-50 nM) of Ncs for 24 h. Cell lysates were then subjected to immunoblotting analysis using the indicated antibodies. Expression levels of p-EGFR, p-Src, and p-STAT3 were quantified and normalized to total EGFR, total Src, and total STAT3 expression levels, respectively. Data were presented as mean values from three independent experiments and error bars represent standard deviations of the mean of three measurements. Magnification: x 100, n.s. >0.05, *P < 0.05, **P < 0.01

    Journal: Journal of Translational Medicine

    Article Title: Narciclasine as a potential therapeutic agent to overcome EGFR-TKI resistance in non-small cell lung cancer

    doi: 10.1186/s12967-025-07368-4

    Figure Lengend Snippet: Effects of Ncs on cell growth in PC-9 and PC-9-GR cells. ( A ) PC-9 and PC-9-GR cells (3 × 10 3 cells/well) were plated in 96-well plates and treated with gefitinib in a dose-dependent manner for 48 h. ( B ) Cells were treated with Ncs at the indicated concentrations for 48 hours, and cell growth rates were measured using the WST-1 assay. ( C ) Cells were treated with Ncs for 48 hours, and representative images are shown. ( D ) Cells (1 × 10 4 cells/well) were plated in 24-well plates and treated with Ncs at the indicated concentrations for 48 h. Live and dead cells were counted using the trypan blue staining assay. ( E and F ) Cells were treated with Ncs for 48 h, fixed with 70% ethanol, stained with PI, and analyzed by flow cytometry to assess cell cycle distribution. ( G ) PC-9 and PC-9-GR cells were treated with the indicated concentrations (0-50 nM) of Ncs for 24 h. Cell lysates were then subjected to immunoblotting analysis using the indicated antibodies. Expression levels of p-EGFR, p-Src, and p-STAT3 were quantified and normalized to total EGFR, total Src, and total STAT3 expression levels, respectively. Data were presented as mean values from three independent experiments and error bars represent standard deviations of the mean of three measurements. Magnification: x 100, n.s. >0.05, *P < 0.05, **P < 0.01

    Article Snippet: Primary antibodies against total EGFR (#4267, Cell Signaling Technology(CST)), CDK1 (#9116, CST), phospho-EGFR Y1068 (#44-788 G, Invitrogen), total STAT3 (#4904, CST), phospho-STAT3 Y705 (#9145, CST), phospho-Src (#6943,CST), total-Src (#2109, CST), phospho-CDK1 Y15 (#4539, CST), phospho-Wee1 (#4910, CST), Myt1 (#4282, CST), p21 (#2947, CST), Cdc25B (#9525, CST), GAPDH (#5174, CST), β-actin (#A700-057, Bethyl Laboratory), phospho-ERK1/2 (#9101, CST), Bcl-2 (#sc-7382, Santa Cruz), and c-Myc (#sc-789, Santa Cruz) were used for immunoblotting.

    Techniques: WST-1 Assay, Staining, Flow Cytometry, Western Blot, Expressing

    Effects of Ncs on EGFR activity and EGFR signaling. ( A ) H1975 cells were treated with 50 nM of Ncs for indicated times and cell lysates were subjected to immunoblotting analysis using the indicated antibodies. ( B ) Cells were treated with 50 nM of Ncs for the indicated durations, and total RNA was isolated. EGFR mRNA levels were determined by real-time PCR analysis and normalized to GAPDH expression. ( C ) Cells were treated with 20 μM of cycloheximide (CHX) for the indicated time points in the presence of either DMSO or 50 nM of Ncs for 24 h. Cell lysates were subjected to immunoblotting analysis using the indicated antibodies (upper panel). EGFR expression levels were quantified from immunoblot bands and normalized to GAPDH, comparing CHX treatment in the presence of DMSO or Ncs (lower panel). ( D ) Cells were treated with 50 nM of Ncs for 24 h in the presence of BafA1 (20 nM, 12 h) or MG132 (20 μM, 2 h). Cell lysates were subjected to immunoblotting analysis using indicated antibodies. ( E ) Cells were plated on coverslips and treated with DMSO (control) or 50 nM of Ncs for 24 h. Cells were then fixed with 4% paraformaldehyde (PFA) and subjected to immunofluorescence staining using DAPI, EGFR, and LAMP1 antibody. ( F ) Cells were treated with 50 nM of Ncs for 24 h, followed by EGF (10 nM, 15 min) stimulation. Cell lysates were subjected to immunoblotting analysis using the specified antibodies. Similar results were observed in three independent experiments. Error bars represent standard deviations of the mean of three measurements, *P < 0.05, **P < 0.01, ***P < 0.001

    Journal: Journal of Translational Medicine

    Article Title: Narciclasine as a potential therapeutic agent to overcome EGFR-TKI resistance in non-small cell lung cancer

    doi: 10.1186/s12967-025-07368-4

    Figure Lengend Snippet: Effects of Ncs on EGFR activity and EGFR signaling. ( A ) H1975 cells were treated with 50 nM of Ncs for indicated times and cell lysates were subjected to immunoblotting analysis using the indicated antibodies. ( B ) Cells were treated with 50 nM of Ncs for the indicated durations, and total RNA was isolated. EGFR mRNA levels were determined by real-time PCR analysis and normalized to GAPDH expression. ( C ) Cells were treated with 20 μM of cycloheximide (CHX) for the indicated time points in the presence of either DMSO or 50 nM of Ncs for 24 h. Cell lysates were subjected to immunoblotting analysis using the indicated antibodies (upper panel). EGFR expression levels were quantified from immunoblot bands and normalized to GAPDH, comparing CHX treatment in the presence of DMSO or Ncs (lower panel). ( D ) Cells were treated with 50 nM of Ncs for 24 h in the presence of BafA1 (20 nM, 12 h) or MG132 (20 μM, 2 h). Cell lysates were subjected to immunoblotting analysis using indicated antibodies. ( E ) Cells were plated on coverslips and treated with DMSO (control) or 50 nM of Ncs for 24 h. Cells were then fixed with 4% paraformaldehyde (PFA) and subjected to immunofluorescence staining using DAPI, EGFR, and LAMP1 antibody. ( F ) Cells were treated with 50 nM of Ncs for 24 h, followed by EGF (10 nM, 15 min) stimulation. Cell lysates were subjected to immunoblotting analysis using the specified antibodies. Similar results were observed in three independent experiments. Error bars represent standard deviations of the mean of three measurements, *P < 0.05, **P < 0.01, ***P < 0.001

    Article Snippet: Primary antibodies against total EGFR (#4267, Cell Signaling Technology(CST)), CDK1 (#9116, CST), phospho-EGFR Y1068 (#44-788 G, Invitrogen), total STAT3 (#4904, CST), phospho-STAT3 Y705 (#9145, CST), phospho-Src (#6943,CST), total-Src (#2109, CST), phospho-CDK1 Y15 (#4539, CST), phospho-Wee1 (#4910, CST), Myt1 (#4282, CST), p21 (#2947, CST), Cdc25B (#9525, CST), GAPDH (#5174, CST), β-actin (#A700-057, Bethyl Laboratory), phospho-ERK1/2 (#9101, CST), Bcl-2 (#sc-7382, Santa Cruz), and c-Myc (#sc-789, Santa Cruz) were used for immunoblotting.

    Techniques: Activity Assay, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Control, Immunofluorescence, Staining

    Anti-cancer effects of ncs in vivo. ( A ) Adult-stage C. elegans models: wild type (WT) and jgIs25 strains. The jgIs25 strain exhibits polyps on the ventral side (arrows in the lower panel). Scale bar = 20 μm. ( B ) Effects of gefitinib and ncs treatment on jgIs25 . the arrows in the upper panel indicate polyps, and the rectangular area is magnified in the lower panel. Scale bar = 200 μm (upper), 20 μm (lower). ( C ) Quantification of polyp formation in jgIs25 following treatment with the indicated drugs. ( D and G ) In vivo tumor model using subcutaneous transplantation of A549 and H1975 cells into nude mice, followed by ncs treatment. Tumor volumes were measured at the indicated time points in control and ncs-treated mice. Tumor weights were measured after sacrifice. ( E and H ) Representative image of xenograft tumors after sacrifice. ( F and I ) Immunohistochemical (IHC) staining of xenograft tumors using anti-EGFR, anti-phospho-EGFR (Y1068), and anti-phospho-STAT3 (Y705) antibodies. Scale bar = 500 μm. Similar results were observed in three independent experiments. Error bars represent standard deviations of the mean of three measurements, *p < 0.05, **p < 0.01, *p < 0.001

    Journal: Journal of Translational Medicine

    Article Title: Narciclasine as a potential therapeutic agent to overcome EGFR-TKI resistance in non-small cell lung cancer

    doi: 10.1186/s12967-025-07368-4

    Figure Lengend Snippet: Anti-cancer effects of ncs in vivo. ( A ) Adult-stage C. elegans models: wild type (WT) and jgIs25 strains. The jgIs25 strain exhibits polyps on the ventral side (arrows in the lower panel). Scale bar = 20 μm. ( B ) Effects of gefitinib and ncs treatment on jgIs25 . the arrows in the upper panel indicate polyps, and the rectangular area is magnified in the lower panel. Scale bar = 200 μm (upper), 20 μm (lower). ( C ) Quantification of polyp formation in jgIs25 following treatment with the indicated drugs. ( D and G ) In vivo tumor model using subcutaneous transplantation of A549 and H1975 cells into nude mice, followed by ncs treatment. Tumor volumes were measured at the indicated time points in control and ncs-treated mice. Tumor weights were measured after sacrifice. ( E and H ) Representative image of xenograft tumors after sacrifice. ( F and I ) Immunohistochemical (IHC) staining of xenograft tumors using anti-EGFR, anti-phospho-EGFR (Y1068), and anti-phospho-STAT3 (Y705) antibodies. Scale bar = 500 μm. Similar results were observed in three independent experiments. Error bars represent standard deviations of the mean of three measurements, *p < 0.05, **p < 0.01, *p < 0.001

    Article Snippet: Primary antibodies against total EGFR (#4267, Cell Signaling Technology(CST)), CDK1 (#9116, CST), phospho-EGFR Y1068 (#44-788 G, Invitrogen), total STAT3 (#4904, CST), phospho-STAT3 Y705 (#9145, CST), phospho-Src (#6943,CST), total-Src (#2109, CST), phospho-CDK1 Y15 (#4539, CST), phospho-Wee1 (#4910, CST), Myt1 (#4282, CST), p21 (#2947, CST), Cdc25B (#9525, CST), GAPDH (#5174, CST), β-actin (#A700-057, Bethyl Laboratory), phospho-ERK1/2 (#9101, CST), Bcl-2 (#sc-7382, Santa Cruz), and c-Myc (#sc-789, Santa Cruz) were used for immunoblotting.

    Techniques: In Vivo, Transplantation Assay, Control, Immunohistochemical staining, Immunohistochemistry

    Matuzumab inhibits EGFR and HER2 phosphorylation, but not Akt and MAPK phosphorylation elicited by EGF . Effects of Matuzumab (100 μg/mL) on EGF-induced activation of EGFR (Tyr 845, 992, 1045 and 1068), HER-2/ neu , Akt and ERK 1/2 on A431, Caski and C33A cells, detected by Western blotting.

    Journal: Molecular Cancer

    Article Title: Efficient Blockade of Akt signaling is a determinant factor to overcome resistance to Matuzumab

    doi: 10.1186/1476-4598-10-151

    Figure Lengend Snippet: Matuzumab inhibits EGFR and HER2 phosphorylation, but not Akt and MAPK phosphorylation elicited by EGF . Effects of Matuzumab (100 μg/mL) on EGF-induced activation of EGFR (Tyr 845, 992, 1045 and 1068), HER-2/ neu , Akt and ERK 1/2 on A431, Caski and C33A cells, detected by Western blotting.

    Article Snippet: Primary antibodies against total and phosphorylated EGFR, HER2, Akt and MAPK (all from Cell Signaling Technology, Beverly, MA, USA) were used.

    Techniques: Activation Assay, Western Blot

    Differences between matuzumab and cetuximab regarding the modulation of EGFR down-regulation, cell proliferation and survival . (A) A431 and Caski cells were incubated with matuzumab or cetuximab (100 μg/mL, each) for 24 h in serum-free culture medium and cells were then lysed for Western blotting analysis of total EGFR. (B) Effects of matuzumab or cetuximab (100 μg/mL, each) on cell metabolic viability (MTT assay) and colony formation (clonogenic assay) of A431, Caski and C33A cells. Student's t test * P < 0.05, when compared to control cells. (C) Effects of MG132 (15 μM), a proteassomal inhibitor, in combination with cetuximab or matuzumab (100 μg/mL, each) on EGFR expression of A431 cells, detected by Western blotting. (D) Effects of MG132 (15 μM) in combination with cetuximab (100 μg/mL) on phosphorylation of EGFR (Tyr 1068), Akt and ERK1/2 on A431 and Caski cells, detected by Western blotting.

    Journal: Molecular Cancer

    Article Title: Efficient Blockade of Akt signaling is a determinant factor to overcome resistance to Matuzumab

    doi: 10.1186/1476-4598-10-151

    Figure Lengend Snippet: Differences between matuzumab and cetuximab regarding the modulation of EGFR down-regulation, cell proliferation and survival . (A) A431 and Caski cells were incubated with matuzumab or cetuximab (100 μg/mL, each) for 24 h in serum-free culture medium and cells were then lysed for Western blotting analysis of total EGFR. (B) Effects of matuzumab or cetuximab (100 μg/mL, each) on cell metabolic viability (MTT assay) and colony formation (clonogenic assay) of A431, Caski and C33A cells. Student's t test * P < 0.05, when compared to control cells. (C) Effects of MG132 (15 μM), a proteassomal inhibitor, in combination with cetuximab or matuzumab (100 μg/mL, each) on EGFR expression of A431 cells, detected by Western blotting. (D) Effects of MG132 (15 μM) in combination with cetuximab (100 μg/mL) on phosphorylation of EGFR (Tyr 1068), Akt and ERK1/2 on A431 and Caski cells, detected by Western blotting.

    Article Snippet: Primary antibodies against total and phosphorylated EGFR, HER2, Akt and MAPK (all from Cell Signaling Technology, Beverly, MA, USA) were used.

    Techniques: Incubation, Western Blot, MTT Assay, Clonogenic Assay, Expressing

    Targeting PAR2 enhanced gefitinib inhibition in ERK phosphorylation and EMT. (A) . Protein expressions of total or phosph-ERK, E-cadherin and vimentin were increased in PC-9-GR cells; (B–C) . P2pal-18S promoted gefitinib to inhibit phosphorylation of ERK in PC-9 (B) and PC-9-GR cells (C) ; (D) . EMT-related gene expressions were detected and compared in gefitinib-sensitive and -resistant cells; (E) . The morphology of PC-9 and PC-9-GR cells was observed; (F–J) . P2pal-18S facilitated gefitinib to increase expressions of E-cadherin and decrease expressions of vimentin; (K) . The heat map of gene expressions of vimentin, E-cadherin, Slug, Bcl-2 and Bax by different treatments compared with control group (value = 1) in PC-9 and PC-9-GR cells, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: Frontiers in Pharmacology

    Article Title: Targeting PAR2 Overcomes Gefitinib Resistance in Non-Small-Cell Lung Cancer Cells Through Inhibition of EGFR Transactivation

    doi: 10.3389/fphar.2021.625289

    Figure Lengend Snippet: Targeting PAR2 enhanced gefitinib inhibition in ERK phosphorylation and EMT. (A) . Protein expressions of total or phosph-ERK, E-cadherin and vimentin were increased in PC-9-GR cells; (B–C) . P2pal-18S promoted gefitinib to inhibit phosphorylation of ERK in PC-9 (B) and PC-9-GR cells (C) ; (D) . EMT-related gene expressions were detected and compared in gefitinib-sensitive and -resistant cells; (E) . The morphology of PC-9 and PC-9-GR cells was observed; (F–J) . P2pal-18S facilitated gefitinib to increase expressions of E-cadherin and decrease expressions of vimentin; (K) . The heat map of gene expressions of vimentin, E-cadherin, Slug, Bcl-2 and Bax by different treatments compared with control group (value = 1) in PC-9 and PC-9-GR cells, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: The primary antibodies used against PAR2, total or phospho-EGFR, total or phospho-ERK as well as the secondary antibodies, HRP-conjugated anti-rabbit IgG, were purchased from Cell Signaling Technology (Boston, MA, United States).

    Techniques: Inhibition, Phospho-proteomics, Control

    PAR2 reversed NSCLC cell resistant to gefitinib via β-arrestin-EGFR-ERK signaling. (A–D) . A β-arrestin inhibitor (Barbadin, 30 μM) facilitated gefitinib to block phosphorylation of EGFR and ERK in PC-9 (A, C) and PC-9-GR cells (B, D) ; (E–F) . Either barbardin or ERK inhibitor (FR180204, 10 μM) largely enhanced inhibitory effects of gefitinib in cell viability of PC-9 (E) and PC-9-GR (F) ; (G–H) . Barbadin or FR180204 also promoted gefitinib to attenuate cell migration of PC-9 (G) and PC-9-GR (H) ; (I–L) . Addition of FR180204 assisted gefitinib to up-regulate or down-regulate expressions of E-cadheirn, vimentin, bcl-2 and bax, suggesting β-arrestin and ERK related to PAR2-mediated EMT and apoptosis (I, K) . The bcl-2/bax ratios were also calculated (J, L) ; (M–P) . Barbadin also enhacanced gefitinib to modulate EMT- or apoptosis-related gene expressions in PC-9 (M) and PC-9-GR cells (O) , while bcl-2/bax ratios were presented (M,P) . * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: Frontiers in Pharmacology

    Article Title: Targeting PAR2 Overcomes Gefitinib Resistance in Non-Small-Cell Lung Cancer Cells Through Inhibition of EGFR Transactivation

    doi: 10.3389/fphar.2021.625289

    Figure Lengend Snippet: PAR2 reversed NSCLC cell resistant to gefitinib via β-arrestin-EGFR-ERK signaling. (A–D) . A β-arrestin inhibitor (Barbadin, 30 μM) facilitated gefitinib to block phosphorylation of EGFR and ERK in PC-9 (A, C) and PC-9-GR cells (B, D) ; (E–F) . Either barbardin or ERK inhibitor (FR180204, 10 μM) largely enhanced inhibitory effects of gefitinib in cell viability of PC-9 (E) and PC-9-GR (F) ; (G–H) . Barbadin or FR180204 also promoted gefitinib to attenuate cell migration of PC-9 (G) and PC-9-GR (H) ; (I–L) . Addition of FR180204 assisted gefitinib to up-regulate or down-regulate expressions of E-cadheirn, vimentin, bcl-2 and bax, suggesting β-arrestin and ERK related to PAR2-mediated EMT and apoptosis (I, K) . The bcl-2/bax ratios were also calculated (J, L) ; (M–P) . Barbadin also enhacanced gefitinib to modulate EMT- or apoptosis-related gene expressions in PC-9 (M) and PC-9-GR cells (O) , while bcl-2/bax ratios were presented (M,P) . * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: The primary antibodies used against PAR2, total or phospho-EGFR, total or phospho-ERK as well as the secondary antibodies, HRP-conjugated anti-rabbit IgG, were purchased from Cell Signaling Technology (Boston, MA, United States).

    Techniques: Blocking Assay, Phospho-proteomics, Migration

    PAR2 inhibition reactivated gefitinib to inhibit tumor growth via ERK signaling in vivo . (A) . The combination of P2pal-18S and gefitinib significantly attenuated PC-9-GR tumor growth; (B) . P2pal-18S promoted gefitinib to decrease tumor growth whereas gefitinib exhibited little inhibitory effects; (C) . The H&E staining analysis of tumor tissues with different treatments were detected; (D) . P2pal-18S facilitated gefitinib to inhibit phosphorylation of ERK in PC-9-GR tumor tissues. Tumor cell necrosis (green), blood capillary (yellow), neutrophil (blue), *** p < 0.001.

    Journal: Frontiers in Pharmacology

    Article Title: Targeting PAR2 Overcomes Gefitinib Resistance in Non-Small-Cell Lung Cancer Cells Through Inhibition of EGFR Transactivation

    doi: 10.3389/fphar.2021.625289

    Figure Lengend Snippet: PAR2 inhibition reactivated gefitinib to inhibit tumor growth via ERK signaling in vivo . (A) . The combination of P2pal-18S and gefitinib significantly attenuated PC-9-GR tumor growth; (B) . P2pal-18S promoted gefitinib to decrease tumor growth whereas gefitinib exhibited little inhibitory effects; (C) . The H&E staining analysis of tumor tissues with different treatments were detected; (D) . P2pal-18S facilitated gefitinib to inhibit phosphorylation of ERK in PC-9-GR tumor tissues. Tumor cell necrosis (green), blood capillary (yellow), neutrophil (blue), *** p < 0.001.

    Article Snippet: The primary antibodies used against PAR2, total or phospho-EGFR, total or phospho-ERK as well as the secondary antibodies, HRP-conjugated anti-rabbit IgG, were purchased from Cell Signaling Technology (Boston, MA, United States).

    Techniques: Inhibition, In Vivo, Staining, Phospho-proteomics

    Tyrosine 1068 phosphorylation in ErbB1. Cerebellar granule cells were grown on the indicated substrates with and without PD168393 at the indicated concentrations and harvested after 16 h. Transfer membranes were probed with an antibody specific for ErbB1 phosphorylated on Y1068. ErbB1 kinase activity was increased by the presence of CNS myelin or fibrinogen but was minimal when PD168393 was added.

    Journal: Experimental Neurology

    Article Title: ErbB1 epidermal growth factor receptor is a valid target for reducing the effects of multiple inhibitors of axonal regeneration

    doi: 10.1016/j.expneurol.2012.09.007

    Figure Lengend Snippet: Tyrosine 1068 phosphorylation in ErbB1. Cerebellar granule cells were grown on the indicated substrates with and without PD168393 at the indicated concentrations and harvested after 16 h. Transfer membranes were probed with an antibody specific for ErbB1 phosphorylated on Y1068. ErbB1 kinase activity was increased by the presence of CNS myelin or fibrinogen but was minimal when PD168393 was added.

    Article Snippet: Immunodetection used rabbit primary antibodies against total and Y1068 phosphorylated EGFR (1:1000, Cell Signaling, Beverly, Massachussetts) together with monoclonal anti-tubulin (1:3000, Sigma).

    Techniques: Phospho-proteomics, Activity Assay

    The TLR3 agonist poly I:C inhibits outgrowth by an ErbB1- and calcium-dependent mechanism. A: Heterozygous and ErbB1 −/− cerebellar granule neurons were cultured on a control substrate in the presence of PD168393 and/or poly I:C at the indicated concentrations. N = 8. Two way ANOVA post hoc Bonferroni, ** = p < 0.01 * = p < 0.05 when compared to heterozygous cells under the same conditions unless shown otherwise, ## = p < 0.01 # = p < 0.05 when compared to genetically identical cells under control conditions. B: Representative images of cultured cerebellar granule neurons from an ErbB1 −/− pup and heterozygote littermates on polylysine/laminin substrates with or without poly I:C then fixed and stained for neuron-specific β3 tubulin. The ErbB1 kinase inhibitor PD168393 was present at 10 nM where indicated. Images were taken at × 20 magnification and scale bar equals 100 μm. The scale bar applies to all panels. C: Heterozygous and ErbB1 −/− sensory neurons were cultured on a control substrate in the presence of PD168393 and/or poly I:C at the indicated concentrations. Neurite length was normalised to the control heterozygous measurement. N = 5. Two way ANOVA post hoc Bonferroni ** = p < 0.01 * = p < 0.05 when compared to heterozygous cells under the same culture conditions unless shown otherwise, ## = p < 0.01 when compared to genetically identical cells under control conditions. D: Representative images of cultured sensory neurons from an ErbB1 −/− pup and heterozygote littermates on polylysine/laminin substrates with or without poly I:C fixed and stained for neuron-specific β3 tubulin. The ErbB1 kinase inhibitor PD168393 was present at 10 nM where indicated. Images were taken at × 20 magnification and scale bar equals 100 μm. The scale bar applies to all panels. E: Heterozygous and ErbB1 −/− sensory neurons were cultured on a control substrate in the presence of PD168393 and/or BCECF AM or BAPTA AM at at the indicated concentrations. Neurite length was normalised to the control heterozygous measurement. N = 5. Two way ANOVA post hoc Bonferroni * = p < 0.05, # = p < 0.05 when compared to genetically identical cells under control conditions. F: Representative images of cultured sensory neurons from an ErbB1 −/− pup and heterozygote littermates on polylysine/laminin substrates in the presence of PD168393 and/or BCECF AM or BAPTA AM at 10 nM, fixed and stained for neuron-specific β3 tubulin. Images were taken at × 20 magnification and scale bar equals 100 μm. The scale bar applies to all panels. G: Western blot showing the effects of 16 h treatment with Poly I:C, PD168393 and BAPTA on ErbB1 kinase activity in cultured cerebellar granule cells. Transfer membranes were probed with an antibody specific for ErbB1 phosphorylated on Y1068.

    Journal: Experimental Neurology

    Article Title: ErbB1 epidermal growth factor receptor is a valid target for reducing the effects of multiple inhibitors of axonal regeneration

    doi: 10.1016/j.expneurol.2012.09.007

    Figure Lengend Snippet: The TLR3 agonist poly I:C inhibits outgrowth by an ErbB1- and calcium-dependent mechanism. A: Heterozygous and ErbB1 −/− cerebellar granule neurons were cultured on a control substrate in the presence of PD168393 and/or poly I:C at the indicated concentrations. N = 8. Two way ANOVA post hoc Bonferroni, ** = p < 0.01 * = p < 0.05 when compared to heterozygous cells under the same conditions unless shown otherwise, ## = p < 0.01 # = p < 0.05 when compared to genetically identical cells under control conditions. B: Representative images of cultured cerebellar granule neurons from an ErbB1 −/− pup and heterozygote littermates on polylysine/laminin substrates with or without poly I:C then fixed and stained for neuron-specific β3 tubulin. The ErbB1 kinase inhibitor PD168393 was present at 10 nM where indicated. Images were taken at × 20 magnification and scale bar equals 100 μm. The scale bar applies to all panels. C: Heterozygous and ErbB1 −/− sensory neurons were cultured on a control substrate in the presence of PD168393 and/or poly I:C at the indicated concentrations. Neurite length was normalised to the control heterozygous measurement. N = 5. Two way ANOVA post hoc Bonferroni ** = p < 0.01 * = p < 0.05 when compared to heterozygous cells under the same culture conditions unless shown otherwise, ## = p < 0.01 when compared to genetically identical cells under control conditions. D: Representative images of cultured sensory neurons from an ErbB1 −/− pup and heterozygote littermates on polylysine/laminin substrates with or without poly I:C fixed and stained for neuron-specific β3 tubulin. The ErbB1 kinase inhibitor PD168393 was present at 10 nM where indicated. Images were taken at × 20 magnification and scale bar equals 100 μm. The scale bar applies to all panels. E: Heterozygous and ErbB1 −/− sensory neurons were cultured on a control substrate in the presence of PD168393 and/or BCECF AM or BAPTA AM at at the indicated concentrations. Neurite length was normalised to the control heterozygous measurement. N = 5. Two way ANOVA post hoc Bonferroni * = p < 0.05, # = p < 0.05 when compared to genetically identical cells under control conditions. F: Representative images of cultured sensory neurons from an ErbB1 −/− pup and heterozygote littermates on polylysine/laminin substrates in the presence of PD168393 and/or BCECF AM or BAPTA AM at 10 nM, fixed and stained for neuron-specific β3 tubulin. Images were taken at × 20 magnification and scale bar equals 100 μm. The scale bar applies to all panels. G: Western blot showing the effects of 16 h treatment with Poly I:C, PD168393 and BAPTA on ErbB1 kinase activity in cultured cerebellar granule cells. Transfer membranes were probed with an antibody specific for ErbB1 phosphorylated on Y1068.

    Article Snippet: Immunodetection used rabbit primary antibodies against total and Y1068 phosphorylated EGFR (1:1000, Cell Signaling, Beverly, Massachussetts) together with monoclonal anti-tubulin (1:3000, Sigma).

    Techniques: Cell Culture, Control, Staining, Western Blot, Activity Assay

    The EGFR/MAPK/ERK1/2 signaling pathway . We have previously shown that sulindac metabolites inhibit both MEK1/2 and ERK1/2 activation. The goal of this study was to determine if this inhibition is due to downregulation of the EGFR.

    Journal: Journal of Carcinogenesis

    Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    doi: 10.1186/1477-3163-4-16

    Figure Lengend Snippet: The EGFR/MAPK/ERK1/2 signaling pathway . We have previously shown that sulindac metabolites inhibit both MEK1/2 and ERK1/2 activation. The goal of this study was to determine if this inhibition is due to downregulation of the EGFR.

    Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

    Techniques: Activation Assay, Inhibition

    EGF induces EGFR and ERK1/2 phosphorylation . HT29 human colon cancer cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 48 h before treatment with vehicle (water), 10, or 100 ng/ml EGF. Cells were harvested 10 min after EGF treatment and lysates prepared for (A) Immunoblotting with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, and total ERK1/2; α-tubulin immunoblots of the same lysates served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and pERK1/2 bands (C) normalized for the loading controls. Data represent mean of triplicate samples ± SD; statistical significance is denoted by ***p < 0.001 versus vehicle. Results shown in the figure are representative of 2 separate experiments each with triplicate samples.

    Journal: Journal of Carcinogenesis

    Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    doi: 10.1186/1477-3163-4-16

    Figure Lengend Snippet: EGF induces EGFR and ERK1/2 phosphorylation . HT29 human colon cancer cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 48 h before treatment with vehicle (water), 10, or 100 ng/ml EGF. Cells were harvested 10 min after EGF treatment and lysates prepared for (A) Immunoblotting with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, and total ERK1/2; α-tubulin immunoblots of the same lysates served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and pERK1/2 bands (C) normalized for the loading controls. Data represent mean of triplicate samples ± SD; statistical significance is denoted by ***p < 0.001 versus vehicle. Results shown in the figure are representative of 2 separate experiments each with triplicate samples.

    Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

    Techniques: Phospho-proteomics, Western Blot

    Dose response of sulindac sulfide inhibition of EGFR . HT29 cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 24 h before addition of drug. Cells were then treated with vehicle (0.1% DMSO), 40, 80, 120, or 160 μM sulindac sulfide, drug doses previously shown to induce apoptotic cell death in these cells. Twenty four hours after drug treatment, vehicle or 10 ng/ml EGF was added and cells were harvested 10 min later. (A) Immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and caspase-3; total ERK1/2 immunoblots served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Results shown in figure are representative of 3 separate experiments.

    Journal: Journal of Carcinogenesis

    Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    doi: 10.1186/1477-3163-4-16

    Figure Lengend Snippet: Dose response of sulindac sulfide inhibition of EGFR . HT29 cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 24 h before addition of drug. Cells were then treated with vehicle (0.1% DMSO), 40, 80, 120, or 160 μM sulindac sulfide, drug doses previously shown to induce apoptotic cell death in these cells. Twenty four hours after drug treatment, vehicle or 10 ng/ml EGF was added and cells were harvested 10 min later. (A) Immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and caspase-3; total ERK1/2 immunoblots served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Results shown in figure are representative of 3 separate experiments.

    Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

    Techniques: Inhibition, Western Blot

    Dose response of sulindac sulfone inhibition of EGFR . HT29 cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 24 h before addition of drug. Cells were then treated with vehicle (0.2% DMSO), 200, 400, 600, or 800 μM sulindac sulfone, drug doses previously shown to induce apoptotic cell death in these cells. Twenty four hours after drug treatment, vehicle or 10 ng/ml EGF was added and cells were harvested 10 min later. (A) Immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and caspase-3; total ERK1/2 immunoblots served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Results shown in figure are representative of 3 separate experiments.

    Journal: Journal of Carcinogenesis

    Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    doi: 10.1186/1477-3163-4-16

    Figure Lengend Snippet: Dose response of sulindac sulfone inhibition of EGFR . HT29 cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 24 h before addition of drug. Cells were then treated with vehicle (0.2% DMSO), 200, 400, 600, or 800 μM sulindac sulfone, drug doses previously shown to induce apoptotic cell death in these cells. Twenty four hours after drug treatment, vehicle or 10 ng/ml EGF was added and cells were harvested 10 min later. (A) Immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and caspase-3; total ERK1/2 immunoblots served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Results shown in figure are representative of 3 separate experiments.

    Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

    Techniques: Inhibition, Western Blot

    Dose response and time course of sulindac sulfide inhibition of EGFR . HT29 cells were grown to confluence in medium containing 10% FBS and treated with vehicle (0.1% DMSO), 160, or 180 μM sulindac sulfide for 1 h, 12 h, and 24 h. Cells were then harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and cleaved caspase-3; α-tubulin immunoblots of the same lysates served as loading controls. (A) 1 h, 12 h, and 24 h immunoblot results. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Data represent mean of triplicate samples ± SD; statistical significance is denoted by **p < 0.01 and ***p < 0.001 versus respective time point vehicle. Results shown in figure are representative of 2 separate experiments each with triplicate samples.

    Journal: Journal of Carcinogenesis

    Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    doi: 10.1186/1477-3163-4-16

    Figure Lengend Snippet: Dose response and time course of sulindac sulfide inhibition of EGFR . HT29 cells were grown to confluence in medium containing 10% FBS and treated with vehicle (0.1% DMSO), 160, or 180 μM sulindac sulfide for 1 h, 12 h, and 24 h. Cells were then harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and cleaved caspase-3; α-tubulin immunoblots of the same lysates served as loading controls. (A) 1 h, 12 h, and 24 h immunoblot results. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Data represent mean of triplicate samples ± SD; statistical significance is denoted by **p < 0.01 and ***p < 0.001 versus respective time point vehicle. Results shown in figure are representative of 2 separate experiments each with triplicate samples.

    Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

    Techniques: Inhibition, Western Blot

    Dose response and time course of sulindac sulfone inhibition of EGFR . HT29 cells were grown to confluence in medium containing 10% FBS followed by treatment with vehicle (0.2% DMSO), 400, or 600 μM sulindac sulfone for 1 h, 12 h, and 24 h. Cells were then harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and cleaved caspase-3; α-tubulin immunoblots of the same lysates served as loading controls. (A) 1 h, 12 h, and 24 h Western blot results. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Data represent mean of triplicate samples ± SD; statistical significance is denoted by **p < 0.01 and ***p < 0.001 versus respective time point vehicle. Results shown in figure are representative of 2 separate experiments each with triplicate samples.

    Journal: Journal of Carcinogenesis

    Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    doi: 10.1186/1477-3163-4-16

    Figure Lengend Snippet: Dose response and time course of sulindac sulfone inhibition of EGFR . HT29 cells were grown to confluence in medium containing 10% FBS followed by treatment with vehicle (0.2% DMSO), 400, or 600 μM sulindac sulfone for 1 h, 12 h, and 24 h. Cells were then harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and cleaved caspase-3; α-tubulin immunoblots of the same lysates served as loading controls. (A) 1 h, 12 h, and 24 h Western blot results. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Data represent mean of triplicate samples ± SD; statistical significance is denoted by **p < 0.01 and ***p < 0.001 versus respective time point vehicle. Results shown in figure are representative of 2 separate experiments each with triplicate samples.

    Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

    Techniques: Inhibition, Western Blot

    Effect of the caspase inhibitor, ZVAD, on apoptosis and inhibition of EGFR . HT29 colon cancer cells were grown to confluence in medium containing 10% FBS followed by pretreatment with or without 25 μM zvad for 1 h. Cells were then treated with vehicle (0.2% DMSO) or 600 μM sulfone for 48 h. Cells were harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, and cleaved caspase 3; α-tubulin immunoblots of the same lysates served as loading controls. The graphs show morphological apoptosis results (A) 48 h Western immunoblot results (B) and densitometry of the pEGFR bands (C) and total EGFR bands (D) . Data represent mean of triplicate samples ± SD; statistical significance is denoted by *p < 0.05, **p < 0.01 and ***p < 0.001. Results shown in figure are representative of 2 separate experiments each with triplicate samples.

    Journal: Journal of Carcinogenesis

    Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    doi: 10.1186/1477-3163-4-16

    Figure Lengend Snippet: Effect of the caspase inhibitor, ZVAD, on apoptosis and inhibition of EGFR . HT29 colon cancer cells were grown to confluence in medium containing 10% FBS followed by pretreatment with or without 25 μM zvad for 1 h. Cells were then treated with vehicle (0.2% DMSO) or 600 μM sulfone for 48 h. Cells were harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, and cleaved caspase 3; α-tubulin immunoblots of the same lysates served as loading controls. The graphs show morphological apoptosis results (A) 48 h Western immunoblot results (B) and densitometry of the pEGFR bands (C) and total EGFR bands (D) . Data represent mean of triplicate samples ± SD; statistical significance is denoted by *p < 0.05, **p < 0.01 and ***p < 0.001. Results shown in figure are representative of 2 separate experiments each with triplicate samples.

    Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

    Techniques: Inhibition, Western Blot