Structured Review

Santa Cruz Biotechnology donkey anti goat igg
K8/K18 physically interacts with DR5 (A) Cell lysates from MCF7 and T47D cell lines were subjected to immunoprecipitation (IP) with an anti-K8/K18 antibody immobilized to protein A-agarose (lane 2 and 4) or with protein A-agarose alone (lane 1 and 3). IP samples were immunoblotted for DR5. (B) Cells were fixed, permeabilized, stained with anti-DR5 antibody (red) and anti-keratin 8 antibody (green), and analyzed by confocal microscopy. All images were acquired using a 40x objective lens. (C) MCF7 cells were transfected with control siRNA or siRNA against KRT8 for 72 hr. The resultant cells were fixed, permeabilized, and stained with anti-DR5 antibody (red), DAPI (nuclei, blue), and fluorescent phalloidin 488 for actin visualization (green). All images were acquired using a 40x objective lens. (D-F) Flow cytometry analysis of cells transfected with control siRNA or siRNA against KRT8 . The resultant cells were harvested using a non-enzymatic cell dissociation solution. The cell samples were incubated with PE conjugated anti-DR5 antibody <t>(IgG2b)</t> or its corresponding IgG2b isotype as a control. (D) Shown are representative histograms of unstained cells (black dashed line), cells stained with the isotype control (red fill), and cells stained with PE-anti-DR5 antibody (blue outline with no fill). The right shift represents the presence of DR5 on the cell surface membrane. Median PE-anti-DR5 antibody fluorescence intensity for cells transfected with control siRNA and siRNA targeting keratin 8 are shown after subtraction of isotype control median fluorescence intensity in two independent experiments for T47D cells (E) and MCF7 cells (F).
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Images

1) Product Images from "Cytokeratin 8/18 protects breast cancer cell lines from TRAIL-induced apoptosis"

Article Title: Cytokeratin 8/18 protects breast cancer cell lines from TRAIL-induced apoptosis

Journal: Oncotarget

doi: 10.18632/oncotarget.25297

K8/K18 physically interacts with DR5 (A) Cell lysates from MCF7 and T47D cell lines were subjected to immunoprecipitation (IP) with an anti-K8/K18 antibody immobilized to protein A-agarose (lane 2 and 4) or with protein A-agarose alone (lane 1 and 3). IP samples were immunoblotted for DR5. (B) Cells were fixed, permeabilized, stained with anti-DR5 antibody (red) and anti-keratin 8 antibody (green), and analyzed by confocal microscopy. All images were acquired using a 40x objective lens. (C) MCF7 cells were transfected with control siRNA or siRNA against KRT8 for 72 hr. The resultant cells were fixed, permeabilized, and stained with anti-DR5 antibody (red), DAPI (nuclei, blue), and fluorescent phalloidin 488 for actin visualization (green). All images were acquired using a 40x objective lens. (D-F) Flow cytometry analysis of cells transfected with control siRNA or siRNA against KRT8 . The resultant cells were harvested using a non-enzymatic cell dissociation solution. The cell samples were incubated with PE conjugated anti-DR5 antibody (IgG2b) or its corresponding IgG2b isotype as a control. (D) Shown are representative histograms of unstained cells (black dashed line), cells stained with the isotype control (red fill), and cells stained with PE-anti-DR5 antibody (blue outline with no fill). The right shift represents the presence of DR5 on the cell surface membrane. Median PE-anti-DR5 antibody fluorescence intensity for cells transfected with control siRNA and siRNA targeting keratin 8 are shown after subtraction of isotype control median fluorescence intensity in two independent experiments for T47D cells (E) and MCF7 cells (F).
Figure Legend Snippet: K8/K18 physically interacts with DR5 (A) Cell lysates from MCF7 and T47D cell lines were subjected to immunoprecipitation (IP) with an anti-K8/K18 antibody immobilized to protein A-agarose (lane 2 and 4) or with protein A-agarose alone (lane 1 and 3). IP samples were immunoblotted for DR5. (B) Cells were fixed, permeabilized, stained with anti-DR5 antibody (red) and anti-keratin 8 antibody (green), and analyzed by confocal microscopy. All images were acquired using a 40x objective lens. (C) MCF7 cells were transfected with control siRNA or siRNA against KRT8 for 72 hr. The resultant cells were fixed, permeabilized, and stained with anti-DR5 antibody (red), DAPI (nuclei, blue), and fluorescent phalloidin 488 for actin visualization (green). All images were acquired using a 40x objective lens. (D-F) Flow cytometry analysis of cells transfected with control siRNA or siRNA against KRT8 . The resultant cells were harvested using a non-enzymatic cell dissociation solution. The cell samples were incubated with PE conjugated anti-DR5 antibody (IgG2b) or its corresponding IgG2b isotype as a control. (D) Shown are representative histograms of unstained cells (black dashed line), cells stained with the isotype control (red fill), and cells stained with PE-anti-DR5 antibody (blue outline with no fill). The right shift represents the presence of DR5 on the cell surface membrane. Median PE-anti-DR5 antibody fluorescence intensity for cells transfected with control siRNA and siRNA targeting keratin 8 are shown after subtraction of isotype control median fluorescence intensity in two independent experiments for T47D cells (E) and MCF7 cells (F).

Techniques Used: Immunoprecipitation, Staining, Confocal Microscopy, Transfection, Flow Cytometry, Cytometry, Incubation, Fluorescence

2) Product Images from "Prognostic Significance of Erythropoietin in Pancreatic Adenocarcinoma"

Article Title: Prognostic Significance of Erythropoietin in Pancreatic Adenocarcinoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023151

Ectopic sources of Epo in tissues of patients with pancreatic diseases. (A) Remnants of Epo-producing islets in degrading CP-parenchyma. (B) Epo-negativity of tumor cells in primary pancreatic lesion, except for sporadic focal staining observed in intracellular vacuoles of tumor cells (C, arrows and inset). Peripheral capillaries (C, arrowheads) and vasa vasorum (D, arrowheads) of bigger vessels frequently demonstrated Epo positivity in PDAC and CP. (E, F) In liver, cytoplasmic staining of hepatocytes was strong in areas directly bordering Epo-negative metastatic tumor cells and inflammatory infiltrates, but faded away distally, thus pointing to the spatially regulated de novo synthesis and creation of multiple Epo-rich niches. Insets in A, D and E depict negative (isotype IgG) controls; insets in C and F show high-magnification (×630) images of staining patterns in intracellular vacuoles of tumor cells and cytoplasm in hepatocytes.
Figure Legend Snippet: Ectopic sources of Epo in tissues of patients with pancreatic diseases. (A) Remnants of Epo-producing islets in degrading CP-parenchyma. (B) Epo-negativity of tumor cells in primary pancreatic lesion, except for sporadic focal staining observed in intracellular vacuoles of tumor cells (C, arrows and inset). Peripheral capillaries (C, arrowheads) and vasa vasorum (D, arrowheads) of bigger vessels frequently demonstrated Epo positivity in PDAC and CP. (E, F) In liver, cytoplasmic staining of hepatocytes was strong in areas directly bordering Epo-negative metastatic tumor cells and inflammatory infiltrates, but faded away distally, thus pointing to the spatially regulated de novo synthesis and creation of multiple Epo-rich niches. Insets in A, D and E depict negative (isotype IgG) controls; insets in C and F show high-magnification (×630) images of staining patterns in intracellular vacuoles of tumor cells and cytoplasm in hepatocytes.

Techniques Used: Staining

3) Product Images from "Caprine humoral response to Burkholderia pseudomallei antigens during acute melioidosis from aerosol exposure"

Article Title: Caprine humoral response to Burkholderia pseudomallei antigens during acute melioidosis from aerosol exposure

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0006851

Comparison of IgG and IgM Immune diversity across eight infected goats against proteins of Burkholderia pseudomallei MSHR511. The average of antigenic spots detected by each individual antibody isotype is shown for each collection time after aerosol challenge and the paired pre-challenge serum sample; 7, 14, and 21 days. In addition, spots that were detected by IgG and IgM antibodies were averaged with pre- and post-challenge immune diversity. Vertical Bars represent immunoglobulins M (IgM, white bar), and immunoglobulin G (IgG, black bar). Circle represents spots detected by both of IgM and IgG (black circle).
Figure Legend Snippet: Comparison of IgG and IgM Immune diversity across eight infected goats against proteins of Burkholderia pseudomallei MSHR511. The average of antigenic spots detected by each individual antibody isotype is shown for each collection time after aerosol challenge and the paired pre-challenge serum sample; 7, 14, and 21 days. In addition, spots that were detected by IgG and IgM antibodies were averaged with pre- and post-challenge immune diversity. Vertical Bars represent immunoglobulins M (IgM, white bar), and immunoglobulin G (IgG, black bar). Circle represents spots detected by both of IgM and IgG (black circle).

Techniques Used: Infection

A time series of IgG and IgM responses to infection with B . pseudomallei . Goat sera were collected prior to infection and on the day of euthanasia. Immunoreactive proteins with IgG (A) and IgM (B) were determined by western blot analysis and then mapped onto a silver stained gel. The number of immunogenic protein spots detected (n) is provided at the bottom of each image.
Figure Legend Snippet: A time series of IgG and IgM responses to infection with B . pseudomallei . Goat sera were collected prior to infection and on the day of euthanasia. Immunoreactive proteins with IgG (A) and IgM (B) were determined by western blot analysis and then mapped onto a silver stained gel. The number of immunogenic protein spots detected (n) is provided at the bottom of each image.

Techniques Used: Infection, Western Blot, Staining

Goat humoral antibody responses to individual protein and polysaccharide antigens. Goat sera were collected prior to challenge and on days 7, 14 and 21 after infection (goat serum from day 16 was averaged with the day 14 calculations). Goat antibody quantities (μg/ml) was calculated by subtracting pre-challenge from post-challenge ELISA results relative to the commercially purchased purified standards of goat IgG and IgM. (A) PDHD, Dihydrolipoamide dehydrogenase of pyruvate dehydrogenase complex; (B) TPX, Thiol peroxidase; (C) AhpC2, Alkyl hydroperoxide reductase subunit C-like protein; (D) Eno, Enolase; (E) GroEL, Heat shock protein 60 family chaperone; (F) CPS, Capsular polysaccharide; and (G) OPS A, Type A O-polysaccharide.
Figure Legend Snippet: Goat humoral antibody responses to individual protein and polysaccharide antigens. Goat sera were collected prior to challenge and on days 7, 14 and 21 after infection (goat serum from day 16 was averaged with the day 14 calculations). Goat antibody quantities (μg/ml) was calculated by subtracting pre-challenge from post-challenge ELISA results relative to the commercially purchased purified standards of goat IgG and IgM. (A) PDHD, Dihydrolipoamide dehydrogenase of pyruvate dehydrogenase complex; (B) TPX, Thiol peroxidase; (C) AhpC2, Alkyl hydroperoxide reductase subunit C-like protein; (D) Eno, Enolase; (E) GroEL, Heat shock protein 60 family chaperone; (F) CPS, Capsular polysaccharide; and (G) OPS A, Type A O-polysaccharide.

Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Purification

Quantitative goat humoral antibody responses to melioidosis as measured by whole cell lysate (WCL) ELISA and western blots. Goats were challenged by aerosol infection with Burkholderia pseudomallei isolate MSHR511. ELISA and western blot analyses were performed using whole cell lysate (WCL) of cultured MSHR511. Goat sera were collected prior to challenge and on days 7, 14 and 21 after infection. ELISA results (left y-axis) show the mean antibody titer from all goats per sampling day (7, 14, or 21) and are represented by vertical bars for IgG (black bar), and IgM (white bar). The amount of goat antibody in micrograms (μg) was calculated by subtracting pre-challenge from post-challenge ELISA results (before averaging) relative to the commercially purchased purified standards of goat IgG and IgM. The right y-axis shows the total number of antigenic spots in western blots from each goat, represented by small squares for IgG (black square), and IgM (white square). The total numbers of antigenic spots for IgM and IgG are listed in Table 1 .
Figure Legend Snippet: Quantitative goat humoral antibody responses to melioidosis as measured by whole cell lysate (WCL) ELISA and western blots. Goats were challenged by aerosol infection with Burkholderia pseudomallei isolate MSHR511. ELISA and western blot analyses were performed using whole cell lysate (WCL) of cultured MSHR511. Goat sera were collected prior to challenge and on days 7, 14 and 21 after infection. ELISA results (left y-axis) show the mean antibody titer from all goats per sampling day (7, 14, or 21) and are represented by vertical bars for IgG (black bar), and IgM (white bar). The amount of goat antibody in micrograms (μg) was calculated by subtracting pre-challenge from post-challenge ELISA results (before averaging) relative to the commercially purchased purified standards of goat IgG and IgM. The right y-axis shows the total number of antigenic spots in western blots from each goat, represented by small squares for IgG (black square), and IgM (white square). The total numbers of antigenic spots for IgM and IgG are listed in Table 1 .

Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot, Infection, Cell Culture, Sampling, Purification

4) Product Images from "HCN1 and HCN2 Proteins Are Expressed in Cochlear Hair Cells"

Article Title: HCN1 and HCN2 Proteins Are Expressed in Cochlear Hair Cells

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.375832

Immunoprecipitation by primary antibodies to filamin A, HCN2, and fascin-2. Immunoreactivity for filamin A detected by DAB ( A ) and immunofluorescence ( B ) (mouse MAB1678 raised to human filamin A crossing to rat; 1:1,000, clone PM6/317, Chemicon) was localized to stereocilia of both inner hair cells ( long arrows ) and outer hair cells ( short arrows ) in rat cochlea. Scale bars for A and B, 10 μm. C , lane 1, full-length filamin A (detected with filamin A primary antibody 1:1,000) in immunoprecipitation complex arising from the use of anti-filamin A for immunoprecipitation (1:100) from rat brain lysate. This antibody recognizes unprocessed filamin A (270–280 kDa, arrow , as well as 170-, 150-, and 120-kDa cleavage fragments (Chemicon)). Lane 2, negative control with brain lysate, without anti-filamin A immunoprecipitation + beads + primary and secondary antibodies; lane 3, standards. D , additional negative control for C . Immunoprecipitation with mouse IgG as negative control probed with antifilamin A. Lane 1, mouse IgG immunoprecipitation of brain lysate + beads, probed with filamin A primary + donkey anti-mouse secondary. Lane 2, mouse IgG (no immunoprecipitation) + beads, probed with filamin A primary + donkey anti-mouse secondary. Lane 3, denatured mouse IgG electrophoresis, probed with filamin A primary + donkey anti-mouse secondary. No protein was observed corresponding to molecular mass of filamin A ( arrow ). E , Western blot of HCN1 (104 kDa, arrow ) in rat brain lysate (1:50, sc-19706, Santa Cruz Biotechnology), lane 1 ; lane 2 , standards. F , immunoprecipitation by anti-filamin A of full-length HCN1. Lane 1, standards; lane 2 , negative control with brain lysate without anti-filamin A immunoprecipitation + beads + primary and secondary antibodies; lane 3 , HCN1 immunoprecipitated with anti-filamin A ( arrow , 104 kDa), detected with goat anti-mouse HCN1 (carboxyl terminus) which crosses to rat HCN1 sequence (sc-19706 Santa Cruz Biotechnology). G , complex of filamin A and HCN1 also contained protocadherin 15 CD3. Lane 1, standards; lane 2 , full-length protocadherin 15 CD3 (189 kDa, arrow ) immunoprecipitated with anti-filamin A detected with custom primary antibody (1:10,000) ( arrow ). Goat anti-chick IgY-HRP (Santa Cruz Biotechnology) was used as the secondary antibody (1:10,000). Lane 3, negative control without anti-filamin A immunoprecipitation but with brain lysate and protein A beads and primary and secondary antibodies. H , Western blot of HCN2 in rat brain lysate. Lane 1, standards; lane 2 , HCN2 detected with a rabbit polyclonal antibody (1:200, Alomone). Predicted molecular masses of 95 and 127 kDa corresponding to unglycosylated ( arrow ) and glycosylated HCN2, respectively. I , HCN2 is not detected in the complex immunoprecipitated by anti-filamin A. Lane 1, standards; lane 2 , HCN2 bands observed in Western ( H ) are not detected in immunoprecipitated complex. Bands at 170–180 kDa may correspond to protocadherin 15 CD3, given that 5 of 15 aa in the epitope targeted by the HCN2 antibody are identical in protocadherin 15 CD3, and further given that protocadherin 15 CD3 is highly concentrated in the complex immunoprecipitated by anti-filamin A ( G ). J , anti-HCN2 immunoprecipitates HCN1 from rat brain lysate. Lane 1, standards; lane 2, anti-HCN2 primary antibody (1:33, Alomone) immunoprecipitates HCN1 unglycosylated (104 kDa, arrow ) and glycosylated (120 and 130 kDa) forms detected with anti-HCN1 primary antibody (Santa Cruz Biotechnology); lane 3 , negative control for immunoprecipitation with all components except anti-HCN2 antibody for immunoprecipitation (brain lysate and beads). K , anti-HCN2 immunoprecipitation of HCN1 complex does not include protocadherin 15 CD3. Lane 1, standards; lane 2, anti-protocadherin 15 CD3 primary antibody (1:7,500), goat anti-chick secondary antibody (1:5,000); lane 3 , negative control without anti-HCN2 antibody for immunoprecipitation (brain lysate and beads). No bands were evident for either experimental ( lane 2 ) or negative control ( lane 3 ) with goat anti-chick IgY as the secondary antibody. In a second protocol, a bovine anti-chick secondary antibody (1:5,000) detected an ∼170-kDa protein in both experimental and negative control (not illustrated). L , HCN2 binds to fascin-2 by yeast two-hybrid co-transformation. Row 1, HCN2 carboxyl terminus in bait vector pGBKT7 plus fascin-2 in prey vector pGADT7; row 2 , negative control with fascin-2 in prey construct plus empty bait construct; row 3 , negative control with HCN2 in bait construct plus empty prey construct. The co-transformation screening was performed once, and then the desired colony was re-plated onto another selection media in triplicate, with negative controls. The same concentrations of yeast were plated in the grids for rows 1- 3. M , anti-fascin-2 immunoprecipitation of HCN2. Lane 1, standards; lane 2 , 95 kDa ( arrow ) and 127-kDa bands, corresponding to unglycosylated and glycosylated HCN2, respectively; lane 3 negative control without anti-fascin 2 antibody in immunoprecipitation (brain lysate and beads). N , HCN1 is in fascin-2 immunoprecipitation complex along with HCN2. Lane 1, standards; lane 2 , unglycosylated ( arrow ) and glycosylated forms of HCN1 at 120 kDa. Lane 3, goat IgG immunoprecipitation negative control, with beads and all components except anti-fascin-2 for immunoprecipitation. Three or more immunoprecipitation experiments were carried out for each protein in a given complex.
Figure Legend Snippet: Immunoprecipitation by primary antibodies to filamin A, HCN2, and fascin-2. Immunoreactivity for filamin A detected by DAB ( A ) and immunofluorescence ( B ) (mouse MAB1678 raised to human filamin A crossing to rat; 1:1,000, clone PM6/317, Chemicon) was localized to stereocilia of both inner hair cells ( long arrows ) and outer hair cells ( short arrows ) in rat cochlea. Scale bars for A and B, 10 μm. C , lane 1, full-length filamin A (detected with filamin A primary antibody 1:1,000) in immunoprecipitation complex arising from the use of anti-filamin A for immunoprecipitation (1:100) from rat brain lysate. This antibody recognizes unprocessed filamin A (270–280 kDa, arrow , as well as 170-, 150-, and 120-kDa cleavage fragments (Chemicon)). Lane 2, negative control with brain lysate, without anti-filamin A immunoprecipitation + beads + primary and secondary antibodies; lane 3, standards. D , additional negative control for C . Immunoprecipitation with mouse IgG as negative control probed with antifilamin A. Lane 1, mouse IgG immunoprecipitation of brain lysate + beads, probed with filamin A primary + donkey anti-mouse secondary. Lane 2, mouse IgG (no immunoprecipitation) + beads, probed with filamin A primary + donkey anti-mouse secondary. Lane 3, denatured mouse IgG electrophoresis, probed with filamin A primary + donkey anti-mouse secondary. No protein was observed corresponding to molecular mass of filamin A ( arrow ). E , Western blot of HCN1 (104 kDa, arrow ) in rat brain lysate (1:50, sc-19706, Santa Cruz Biotechnology), lane 1 ; lane 2 , standards. F , immunoprecipitation by anti-filamin A of full-length HCN1. Lane 1, standards; lane 2 , negative control with brain lysate without anti-filamin A immunoprecipitation + beads + primary and secondary antibodies; lane 3 , HCN1 immunoprecipitated with anti-filamin A ( arrow , 104 kDa), detected with goat anti-mouse HCN1 (carboxyl terminus) which crosses to rat HCN1 sequence (sc-19706 Santa Cruz Biotechnology). G , complex of filamin A and HCN1 also contained protocadherin 15 CD3. Lane 1, standards; lane 2 , full-length protocadherin 15 CD3 (189 kDa, arrow ) immunoprecipitated with anti-filamin A detected with custom primary antibody (1:10,000) ( arrow ). Goat anti-chick IgY-HRP (Santa Cruz Biotechnology) was used as the secondary antibody (1:10,000). Lane 3, negative control without anti-filamin A immunoprecipitation but with brain lysate and protein A beads and primary and secondary antibodies. H , Western blot of HCN2 in rat brain lysate. Lane 1, standards; lane 2 , HCN2 detected with a rabbit polyclonal antibody (1:200, Alomone). Predicted molecular masses of 95 and 127 kDa corresponding to unglycosylated ( arrow ) and glycosylated HCN2, respectively. I , HCN2 is not detected in the complex immunoprecipitated by anti-filamin A. Lane 1, standards; lane 2 , HCN2 bands observed in Western ( H ) are not detected in immunoprecipitated complex. Bands at 170–180 kDa may correspond to protocadherin 15 CD3, given that 5 of 15 aa in the epitope targeted by the HCN2 antibody are identical in protocadherin 15 CD3, and further given that protocadherin 15 CD3 is highly concentrated in the complex immunoprecipitated by anti-filamin A ( G ). J , anti-HCN2 immunoprecipitates HCN1 from rat brain lysate. Lane 1, standards; lane 2, anti-HCN2 primary antibody (1:33, Alomone) immunoprecipitates HCN1 unglycosylated (104 kDa, arrow ) and glycosylated (120 and 130 kDa) forms detected with anti-HCN1 primary antibody (Santa Cruz Biotechnology); lane 3 , negative control for immunoprecipitation with all components except anti-HCN2 antibody for immunoprecipitation (brain lysate and beads). K , anti-HCN2 immunoprecipitation of HCN1 complex does not include protocadherin 15 CD3. Lane 1, standards; lane 2, anti-protocadherin 15 CD3 primary antibody (1:7,500), goat anti-chick secondary antibody (1:5,000); lane 3 , negative control without anti-HCN2 antibody for immunoprecipitation (brain lysate and beads). No bands were evident for either experimental ( lane 2 ) or negative control ( lane 3 ) with goat anti-chick IgY as the secondary antibody. In a second protocol, a bovine anti-chick secondary antibody (1:5,000) detected an ∼170-kDa protein in both experimental and negative control (not illustrated). L , HCN2 binds to fascin-2 by yeast two-hybrid co-transformation. Row 1, HCN2 carboxyl terminus in bait vector pGBKT7 plus fascin-2 in prey vector pGADT7; row 2 , negative control with fascin-2 in prey construct plus empty bait construct; row 3 , negative control with HCN2 in bait construct plus empty prey construct. The co-transformation screening was performed once, and then the desired colony was re-plated onto another selection media in triplicate, with negative controls. The same concentrations of yeast were plated in the grids for rows 1- 3. M , anti-fascin-2 immunoprecipitation of HCN2. Lane 1, standards; lane 2 , 95 kDa ( arrow ) and 127-kDa bands, corresponding to unglycosylated and glycosylated HCN2, respectively; lane 3 negative control without anti-fascin 2 antibody in immunoprecipitation (brain lysate and beads). N , HCN1 is in fascin-2 immunoprecipitation complex along with HCN2. Lane 1, standards; lane 2 , unglycosylated ( arrow ) and glycosylated forms of HCN1 at 120 kDa. Lane 3, goat IgG immunoprecipitation negative control, with beads and all components except anti-fascin-2 for immunoprecipitation. Three or more immunoprecipitation experiments were carried out for each protein in a given complex.

Techniques Used: Immunoprecipitation, Immunofluorescence, Negative Control, Electrophoresis, Western Blot, Sequencing, Transformation Assay, Plasmid Preparation, Construct, Selection

5) Product Images from "Yes and Lyn play a role in nuclear translocation of the Epidermal Growth Factor Receptor"

Article Title: Yes and Lyn play a role in nuclear translocation of the Epidermal Growth Factor Receptor

Journal: Oncogene

doi: 10.1038/onc.2012.90

Yes and Lyn SFK family members are overexpressed in Ctx R cells (A) Yes and Lyn mRNA is up-regulated in Ctx R clones (HC1, HC4, and HC8). Lyn (3.6~4.5-fold), Yes (~1.5-fold) and Src (−2.3~−3.5-fold) mRNA expression levels were compared to that of the Ctx S cell line (HP) by qPCR. (B) Total protein levels of Yes and Lyn were increased (1.3~2.1-fold) in Ctx R clones (HC1, HC4, and HC8) as compared to the Ctx S cell line (HP). Cells were harvested and protein lysates were fractionated on SDS-PAGE followed by immunoblotting for the indicated proteins. α-tubulin was used as a loading control. Protein expression was quantitated using ImageJ software. (C) Analysis of EGFR binding partners in Ctx R cells using immunoprecipitation assay indicated that EGFR displayed increased binding with Yes and Lyn as compared to the Ctx S parental cell line. Cells were harvested and EGFR or IgG were immunoprecipitated with anti-mouse EGFR antibody or normal mouse IgG. The immunoprecipitate complexes were fractionated on SDS-PAGE followed by immunoblotting for indicated proteins.
Figure Legend Snippet: Yes and Lyn SFK family members are overexpressed in Ctx R cells (A) Yes and Lyn mRNA is up-regulated in Ctx R clones (HC1, HC4, and HC8). Lyn (3.6~4.5-fold), Yes (~1.5-fold) and Src (−2.3~−3.5-fold) mRNA expression levels were compared to that of the Ctx S cell line (HP) by qPCR. (B) Total protein levels of Yes and Lyn were increased (1.3~2.1-fold) in Ctx R clones (HC1, HC4, and HC8) as compared to the Ctx S cell line (HP). Cells were harvested and protein lysates were fractionated on SDS-PAGE followed by immunoblotting for the indicated proteins. α-tubulin was used as a loading control. Protein expression was quantitated using ImageJ software. (C) Analysis of EGFR binding partners in Ctx R cells using immunoprecipitation assay indicated that EGFR displayed increased binding with Yes and Lyn as compared to the Ctx S parental cell line. Cells were harvested and EGFR or IgG were immunoprecipitated with anti-mouse EGFR antibody or normal mouse IgG. The immunoprecipitate complexes were fractionated on SDS-PAGE followed by immunoblotting for indicated proteins.

Techniques Used: Clone Assay, Expressing, Real-time Polymerase Chain Reaction, SDS Page, Software, Binding Assay, Immunoprecipitation

6) Product Images from "Nuclear Epidermal Growth Factor Receptor is a Functional Molecular Target in Triple-Negative Breast Cancer"

Article Title: Nuclear Epidermal Growth Factor Receptor is a Functional Molecular Target in Triple-Negative Breast Cancer

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-13-1021

Therapeutic inhibition of SFKs can block nEGFR translocation in TNBC cell lines and tumor models (A) Dasatinib can inhibit nEGFR translocation and enhance non-nuclear EGFR levels. Cells were treated with vehicle or dasatinib (25 nM) for 24 and 72 hr prior to harvesting whole cell, non-nuclear and nuclear proteins. (B) Dasatinib can block nEGFR translocation measured by Nuance imaging analysis. Cells were treated with vehicle or dasatinib (50 nM) for 24 and 48 hr prior to staining for EGFR, E-Cadherin, and DAPI. Nuclear EGFR fluorescence detected from dasatinib treated cells was normalized to nEGFR fluorescence detected from vehicle treated cells using InForm software (n=2). (C) Dasatinib can enhance plasma membrane-bound EGFR levels measured by flow cytometry. Cells were treated with dasatinib (25 nM) for 24 hr prior to EGFR surface level analysis. Surface level EGFR expression of dasatinib treated cells was normalized to vehicle treated cells (n=3). Shaded histogram= vehicle treated cells, Non-shaded histograms= dasatinib treated cells. IgG treated cells are used as a control (dotted line). (D, E) Dasatinib can block nEGFR translocation in MDAMB468 xenograft tumors. Mice with established MDAMB468 tumors were treated with 50 mg/kg of dasatinib or vehicle once a day for 4 days. Tumors were analyzed by confocal IF (D) and IHC (E) for EGFR expression. IF: merged images were magnified to depict nEGFR (arrows) and non-nEGFR (triangle). Magnification 600X for IF and 400X for IHC. Four tumors from vehicle (tumor # 1–4) or dasatinib treated mice (tumor # 5–8) were harvested for protein and analyzed for the indicated proteins. Data points are represented as mean±s.e.m. **p
Figure Legend Snippet: Therapeutic inhibition of SFKs can block nEGFR translocation in TNBC cell lines and tumor models (A) Dasatinib can inhibit nEGFR translocation and enhance non-nuclear EGFR levels. Cells were treated with vehicle or dasatinib (25 nM) for 24 and 72 hr prior to harvesting whole cell, non-nuclear and nuclear proteins. (B) Dasatinib can block nEGFR translocation measured by Nuance imaging analysis. Cells were treated with vehicle or dasatinib (50 nM) for 24 and 48 hr prior to staining for EGFR, E-Cadherin, and DAPI. Nuclear EGFR fluorescence detected from dasatinib treated cells was normalized to nEGFR fluorescence detected from vehicle treated cells using InForm software (n=2). (C) Dasatinib can enhance plasma membrane-bound EGFR levels measured by flow cytometry. Cells were treated with dasatinib (25 nM) for 24 hr prior to EGFR surface level analysis. Surface level EGFR expression of dasatinib treated cells was normalized to vehicle treated cells (n=3). Shaded histogram= vehicle treated cells, Non-shaded histograms= dasatinib treated cells. IgG treated cells are used as a control (dotted line). (D, E) Dasatinib can block nEGFR translocation in MDAMB468 xenograft tumors. Mice with established MDAMB468 tumors were treated with 50 mg/kg of dasatinib or vehicle once a day for 4 days. Tumors were analyzed by confocal IF (D) and IHC (E) for EGFR expression. IF: merged images were magnified to depict nEGFR (arrows) and non-nEGFR (triangle). Magnification 600X for IF and 400X for IHC. Four tumors from vehicle (tumor # 1–4) or dasatinib treated mice (tumor # 5–8) were harvested for protein and analyzed for the indicated proteins. Data points are represented as mean±s.e.m. **p

Techniques Used: Inhibition, Blocking Assay, Translocation Assay, Imaging, Staining, Fluorescence, Software, Flow Cytometry, Cytometry, Expressing, Mouse Assay, Immunohistochemistry

7) Product Images from "The receptor tyrosine kinase AXL mediates nuclear translocation of the epidermal growth factor receptor"

Article Title: The receptor tyrosine kinase AXL mediates nuclear translocation of the epidermal growth factor receptor

Journal: Science signaling

doi: 10.1126/scisignal.aag1064

Ctx R clones and xenografts have increased abundance of nEGFR and AXL. (A) Immunoblotting (top, left) in non-nuclear and nuclear lysates harvested from three Ctx R clones (HC1, HC4, and HC8) and the Ctx S parental cell line HP. Calnexin, α-Tubulin, and histone H3 were used as loading and purity controls for non-nuclear and nuclear lysates, respectively. Structured resolution microscopy (bottom) in parental (HP) and Ctx R clones stained for DAPI (blue) and EGFR (red) (bottom). Magnification, 100X. Scale bars, 10μm. Transmission electron microscopy (TEM; right) of fixed HC4 cells labeled with EGFR antibody-bound gold particles. Black arrows in insets mark gold particles in the nucleus. Cyto, cytoplasm; NE, nuclear envelope; nPore, nuclear pore; Nuc, nucleus. Scale bars, 0.5 μm. (B) Immunobloting in whole cell lysates from HP and Ctx R clones. GAPDH: loading control. (C) Tumor volume (left) and immunohistochemical staining for EGFR and AXL in representative NCI-H226 xenografts (right) from IgG- or cetuximab-treated mice (n=4 and 5 mice, respectively; representative sections from 3 of each group are shown). Magnification, 40X. Scale bars, 1000 μm. (D) Immunohistochemical staining for EGFR and AXL abundance in Ctx S and Ctx R NSCLC PDXs. Magnification, 20X. Scale bars, 1000 μm. Black arrows in insets mark nEGFR. nEGFR and AXL abundance were quantified with ImageJ software. AXL staining intensity in Ctx R tumors was normalized to that of the IgG or Ctx S tumors (n=6 mice; representative sections from 3 of each group are shown). Data are means ± SD of 3 independent fields of view per tumor. * P
Figure Legend Snippet: Ctx R clones and xenografts have increased abundance of nEGFR and AXL. (A) Immunoblotting (top, left) in non-nuclear and nuclear lysates harvested from three Ctx R clones (HC1, HC4, and HC8) and the Ctx S parental cell line HP. Calnexin, α-Tubulin, and histone H3 were used as loading and purity controls for non-nuclear and nuclear lysates, respectively. Structured resolution microscopy (bottom) in parental (HP) and Ctx R clones stained for DAPI (blue) and EGFR (red) (bottom). Magnification, 100X. Scale bars, 10μm. Transmission electron microscopy (TEM; right) of fixed HC4 cells labeled with EGFR antibody-bound gold particles. Black arrows in insets mark gold particles in the nucleus. Cyto, cytoplasm; NE, nuclear envelope; nPore, nuclear pore; Nuc, nucleus. Scale bars, 0.5 μm. (B) Immunobloting in whole cell lysates from HP and Ctx R clones. GAPDH: loading control. (C) Tumor volume (left) and immunohistochemical staining for EGFR and AXL in representative NCI-H226 xenografts (right) from IgG- or cetuximab-treated mice (n=4 and 5 mice, respectively; representative sections from 3 of each group are shown). Magnification, 40X. Scale bars, 1000 μm. (D) Immunohistochemical staining for EGFR and AXL abundance in Ctx S and Ctx R NSCLC PDXs. Magnification, 20X. Scale bars, 1000 μm. Black arrows in insets mark nEGFR. nEGFR and AXL abundance were quantified with ImageJ software. AXL staining intensity in Ctx R tumors was normalized to that of the IgG or Ctx S tumors (n=6 mice; representative sections from 3 of each group are shown). Data are means ± SD of 3 independent fields of view per tumor. * P

Techniques Used: Clone Assay, Microscopy, Staining, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Labeling, Western Blot, Immunohistochemistry, Mouse Assay, Software

8) Product Images from "Cytokeratin 8/18 protects breast cancer cell lines from TRAIL-induced apoptosis"

Article Title: Cytokeratin 8/18 protects breast cancer cell lines from TRAIL-induced apoptosis

Journal: Oncotarget

doi: 10.18632/oncotarget.25297

K8/K18 physically interacts with DR5 (A) Cell lysates from MCF7 and T47D cell lines were subjected to immunoprecipitation (IP) with an anti-K8/K18 antibody immobilized to protein A-agarose (lane 2 and 4) or with protein A-agarose alone (lane 1 and 3). IP samples were immunoblotted for DR5. (B) Cells were fixed, permeabilized, stained with anti-DR5 antibody (red) and anti-keratin 8 antibody (green), and analyzed by confocal microscopy. All images were acquired using a 40x objective lens. (C) MCF7 cells were transfected with control siRNA or siRNA against KRT8 for 72 hr. The resultant cells were fixed, permeabilized, and stained with anti-DR5 antibody (red), DAPI (nuclei, blue), and fluorescent phalloidin 488 for actin visualization (green). All images were acquired using a 40x objective lens. (D-F) Flow cytometry analysis of cells transfected with control siRNA or siRNA against KRT8 . The resultant cells were harvested using a non-enzymatic cell dissociation solution. The cell samples were incubated with PE conjugated anti-DR5 antibody (IgG2b) or its corresponding IgG2b isotype as a control. (D) Shown are representative histograms of unstained cells (black dashed line), cells stained with the isotype control (red fill), and cells stained with PE-anti-DR5 antibody (blue outline with no fill). The right shift represents the presence of DR5 on the cell surface membrane. Median PE-anti-DR5 antibody fluorescence intensity for cells transfected with control siRNA and siRNA targeting keratin 8 are shown after subtraction of isotype control median fluorescence intensity in two independent experiments for T47D cells (E) and MCF7 cells (F).
Figure Legend Snippet: K8/K18 physically interacts with DR5 (A) Cell lysates from MCF7 and T47D cell lines were subjected to immunoprecipitation (IP) with an anti-K8/K18 antibody immobilized to protein A-agarose (lane 2 and 4) or with protein A-agarose alone (lane 1 and 3). IP samples were immunoblotted for DR5. (B) Cells were fixed, permeabilized, stained with anti-DR5 antibody (red) and anti-keratin 8 antibody (green), and analyzed by confocal microscopy. All images were acquired using a 40x objective lens. (C) MCF7 cells were transfected with control siRNA or siRNA against KRT8 for 72 hr. The resultant cells were fixed, permeabilized, and stained with anti-DR5 antibody (red), DAPI (nuclei, blue), and fluorescent phalloidin 488 for actin visualization (green). All images were acquired using a 40x objective lens. (D-F) Flow cytometry analysis of cells transfected with control siRNA or siRNA against KRT8 . The resultant cells were harvested using a non-enzymatic cell dissociation solution. The cell samples were incubated with PE conjugated anti-DR5 antibody (IgG2b) or its corresponding IgG2b isotype as a control. (D) Shown are representative histograms of unstained cells (black dashed line), cells stained with the isotype control (red fill), and cells stained with PE-anti-DR5 antibody (blue outline with no fill). The right shift represents the presence of DR5 on the cell surface membrane. Median PE-anti-DR5 antibody fluorescence intensity for cells transfected with control siRNA and siRNA targeting keratin 8 are shown after subtraction of isotype control median fluorescence intensity in two independent experiments for T47D cells (E) and MCF7 cells (F).

Techniques Used: Immunoprecipitation, Staining, Confocal Microscopy, Transfection, Flow Cytometry, Cytometry, Incubation, Fluorescence

9) Product Images from "Heteromerization of Ciliary G Protein-Coupled Receptors in the Mouse Brain"

Article Title: Heteromerization of Ciliary G Protein-Coupled Receptors in the Mouse Brain

Journal: PLoS ONE

doi: 10.1371/journal.pone.0046304

Mchr1 and Sstr3 interact in mouse hippocampal lysate. Membrane protein enriched cell lysate from the hippocampus of 5 week old adult WT mice was immunoprecipitated (IP) with a goat anti-Mchr1 antibody or goat IgG, as a negative control. Immunoprecipitates were analyzed by western blotting (IB) with a rabbit anti-Sstr3 antibody. Note that Sstr3 is immunoprecipitated with Mchr1 but not with the IgG negative control. The input probed with anti-Sstr3 (left) or anti-Mchr1 (right), confirms the expression of Sstr3 and Mchr1. The ∼55 kDa bands in the IP lanes may be IgG heavy chain that is detected due to cross-reactivity of the secondary antibody.
Figure Legend Snippet: Mchr1 and Sstr3 interact in mouse hippocampal lysate. Membrane protein enriched cell lysate from the hippocampus of 5 week old adult WT mice was immunoprecipitated (IP) with a goat anti-Mchr1 antibody or goat IgG, as a negative control. Immunoprecipitates were analyzed by western blotting (IB) with a rabbit anti-Sstr3 antibody. Note that Sstr3 is immunoprecipitated with Mchr1 but not with the IgG negative control. The input probed with anti-Sstr3 (left) or anti-Mchr1 (right), confirms the expression of Sstr3 and Mchr1. The ∼55 kDa bands in the IP lanes may be IgG heavy chain that is detected due to cross-reactivity of the secondary antibody.

Techniques Used: Mouse Assay, Immunoprecipitation, Negative Control, Western Blot, Expressing

Related Articles

Immunoprecipitation:

Article Title: The receptor tyrosine kinase AXL mediates nuclear translocation of the epidermal growth factor receptor
Article Snippet: .. Antibodies against EGFR, HER3, AXL (for immunoprecipitation), Histone H3, NRG1, and horse radish peroxidase (HRP)-conjugated goat-anti-rabbit IgG, goat-anti-mouse IgG, donkey-anti-goat IgG were purchased from Santa Cruz Biotechnology Inc.. Antibody against AXL for immunofluorescence was purchased from Life Technologies. .. Antibody against phosphorylated EGFR (Tyr1101 ) was purchased from Abcam, and that against α-Tubulin was purchased from Calbiochem.

Incubation:

Article Title: Prognostic Significance of Erythropoietin in Pancreatic Adenocarcinoma
Article Snippet: .. After overnight incubation at 4°C, the secondary horseradish peroxidase (HRP)-labeled antibodies were applied at room temperature for 45 min (donkey anti-goat IgG for EPO; Santa Cruz Biotechnology/SCBT, Heidelberg, Germany, or polymer-conjugated goat anti-mouse IgG for EpoR; EnVision™+ System, Dako). .. The HRP-reaction product was visualized using a DAB/H2 O2 substrate mixture kit (EnVision™, Dako) and sections were counterstained with Mayer's hematoxylin.

other:

Article Title: Yes and Lyn play a role in nuclear translocation of the Epidermal Growth Factor Receptor
Article Snippet: Antibodies All antibodies were purchased from commercial sources as indicated below: EGFR, B-Myb, Actin, Histone H3, HRP-conjugated goat-anti-rabbit IgG, goat-anti-mouse IgG and donkey-anti-goat IgG were obtained from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA).

Immunofluorescence:

Article Title: The receptor tyrosine kinase AXL mediates nuclear translocation of the epidermal growth factor receptor
Article Snippet: .. Antibodies against EGFR, HER3, AXL (for immunoprecipitation), Histone H3, NRG1, and horse radish peroxidase (HRP)-conjugated goat-anti-rabbit IgG, goat-anti-mouse IgG, donkey-anti-goat IgG were purchased from Santa Cruz Biotechnology Inc.. Antibody against AXL for immunofluorescence was purchased from Life Technologies. .. Antibody against phosphorylated EGFR (Tyr1101 ) was purchased from Abcam, and that against α-Tubulin was purchased from Calbiochem.

Blocking Assay:

Article Title: Nuclear Epidermal Growth Factor Receptor is a Functional Molecular Target in Triple-Negative Breast Cancer
Article Snippet: .. All antibodies were obtained from the following sources: EGFR (SC-03), pEGFR-1173 (SC-10168), HER2 (SC-284), SLAP (SC-1215), Histone H3 (SC-8654), HRP-conjugated goat-anti-rabbit IgG, goat-anti-mouse IgG, donkey-anti-goat IgG, EGFR blocking peptide (SC-03 P) purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). .. SFK (CS2123), pSFK-Y419 (CS2101), pEGFR-Y1045 (CS2237), pEGFR-Y1068 (CS3777), pHER2-Y1221/1222 (CS2243), c-Cbl (CS2747), GAPDH (CS2118), calnexin (CS2679), and anti-Flag (CS8146) purchased from Cell Signaling Technology (Beverly, MA, USA). pEGFR-Y1101 (ab76195) and EGFR (ab52894) purchased from Abcam (Cambridge, MA, USA). α-Tubulin purchased from Calbiochem (San Diego, CA, USA).

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    Santa Cruz Biotechnology tritc conjugated secondary antibody
    Immunofluorescent images showing the deposition of osteocalcin (green, FITC) and osteopontin (red, <t>TRITC)</t> on nanofiber scaffold surfaces. Images obtained on 0% HAp ( A B ), 1% HAp ( C D ), or 10% HAp ( E F ) scaffolds. Magnification is 10×, scale bar is 50 mm in all images.
    Tritc Conjugated Secondary Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology fitc conjugated donkey anti goat igg
    CMTM7 is localized at the plasma membrane in association with clathrin and sIgM. (A) HeLa cells stably expressing T7-hCMTM7 were fixed, permeabilized, and stained with antibodies for the indicated organelle makers (left panels) and T7 tag (middle panels) as described in the Materials and Methods section. Right panels: merged images. (B) HEK293T cells transiently expressing mouse CMTM7 tagged with HA or FLAG epitopes at the N- or C-termini, respectively, or not (schematically represented at the top), were stained with anti-HA antibody (left) or anti-FLAG antibody (right) and analyzed by flow cytometry. The histograms indicate fluorescence intensities for non-tagged (red lines), and HA- (blue lines) or FLAG- (green lines) tagged CMTM7s. The predicted plasma membrane topology of CMTM7 is depicted at the bottom. (C) BAL17 cells stably expressing T7-tagged mouse CMTM7 (BAL17/T7-CMTM7) were stained with rabbit anti-T7 and biotin-anti-IgM antibodies, and incubated at 37°C for the indicated time periods. Then the cells were fixed, permeabilized, and stained with TRITC-anti-rabbit <t>IgG</t> and <t>FITC-streptavidin.</t> The fluorescence FITC (top), TRITC (middle), and their merged images (bottom) are shown.
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    <t>Integrin</t> VLA-3 is a receptor for APC (A) Binding assay of soluble VLA-3, VLA-5, and α V β 3 to APC. Soluble integrins were incubated with the indicated amount of rhAPC in the presence of 1 mM MnCl 2 + integrin activating Abs. The protein complexes were precipitated and subjected to SDS-PAGE and western blotting with an APC Ab (upper panel). The total amounts of soluble integrins precipitated were shown by silver staining (lower panels). (B) rhAPC was incubated with human neutrophils in the presence of 1 mM MnCl 2 . The cells were pretreated with control <t>IgG</t> (Con IgG) or blocking antibodies against integrins VLA-3 (α 3 ), VLA-5 (α 5 ), α V β 3 (β 3 ), and Mac-1 (α M ). Bound APC was detected by flow cytometry. APC binding in the presence of integrin blocking antibodies is expressed as the percentage increase in MFI, compared with the cells not treated with APC (control) in the presence of the same blocking antibody. The data shows the mean ± SEM of three donors. * indicates significance between Con IgG vs. anti-VLA-3 (α 3 ). * p
    Pe Conjugated Donkey Anti Goat Igg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p53 represses the K14 promoter indirectly by associating with transcription factor SP1. (A) The sequence map of the first 160 base pairs of the K14 promoter. No p53 consensus binding site was found within the K14 promoter based on a search for the following typical p53 consensus binding site: PuPuPuC(T/A)(T/A)GPyPyPy-N(013)-PuPuPuC(T/A)(T/A)GpyPyPy; N(0–13) indicates a spacer of 0 to 13 bases. There is an SP1 binding site between base pairs -48 and -43 and a TATA box binding site located between base pairs -29 and -24. The K14 promoter sequence from the GenBank database [GenBank: HSU11076] is shown, and the transcription initiation site refers to the NCBI reference sequence of the human K14 gene [GenBank: NM_000526.4]. (B) H1299 cells were co-transfected with wild-type K14 (pK14WT-160), the TATA site mutant (pK14mtTATA-160), or the SP1 site mutant (pK14mtSP1-160) promoter vectors and pcDNA 3.0 or wild-type p53 expression vectors. The levels of luciferase signal were normalized to the luciferase level of pcDNA 3.0, which was set to 1. p53 was able to repress luciferase expression driven by the pK14WT-160 and pK14mtTATA-160 promoter constructs, but not the pK14mtSP1-160 promoter construct. (C) H1299 cells were co-transfected with pK14-2000 or pK14-160 promoter constructs and pcDNA 3.0, wild-type p53, or p53Δ293-393 expression vectors. The level of luciferase signal from each sample was normalized to the pcDNA 3.0 sample, which was set to 1. p53 but not p53Δ293-393 suppressed luciferase expression driven by the pK14-2000 and pK14-160 promoters. (D) DAPA was used to purify the nuclear protein extract from H1299 cells transfected with the p53Δ363-393 or p53Δ293-393 expression vector. Nuclear proteins were incubated with oligonucleotide probes corresponding to base pairs -53 to -38 of the K14 promoter with a wild-type SP1 binding site CCCGCC or with an SP1 binding site mutant GGTACC. Nuclear proteins that co-precipitated with the probes were blotted with anti-SP1 or anti-p53 antibodies. The SP1 binding site on the K14 promoter co-precipitated with SP1 and p53Δ363-393 but not p53Δ293-393. The SP1 binding site mutant probe was unable to pull down SP1, p53Δ363-393, or p53Δ293-393. “5% input” denotes the positive loading control for nuclear protein, and “beads only” denotes the non-probed negative control. (E) In the ChIP assay, Sp1 and p53 antibodies immunoprecipitated base pair -220 to -5 of the K14 promoter in C9 cells. Non-specific <t>IgG</t> antibody was included as the negative control. IgG, Sp1 or p53 antibody was unable to immunoprecipitate the K14 promoter fragment from base pair -689 to -551. (I: input; G: IgG).
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    Immunofluorescent images showing the deposition of osteocalcin (green, FITC) and osteopontin (red, TRITC) on nanofiber scaffold surfaces. Images obtained on 0% HAp ( A B ), 1% HAp ( C D ), or 10% HAp ( E F ) scaffolds. Magnification is 10×, scale bar is 50 mm in all images.

    Journal: Journal of Functional Biomaterials

    Article Title: Mineralization Content Alters Osteogenic Responses of Bone Marrow Stromal Cells on Hydroxyapatite/Polycaprolactone Composite Nanofiber Scaffolds

    doi: 10.3390/jfb3040776

    Figure Lengend Snippet: Immunofluorescent images showing the deposition of osteocalcin (green, FITC) and osteopontin (red, TRITC) on nanofiber scaffold surfaces. Images obtained on 0% HAp ( A B ), 1% HAp ( C D ), or 10% HAp ( E F ) scaffolds. Magnification is 10×, scale bar is 50 mm in all images.

    Article Snippet: After an additional blocking step and PBS wash, the scaffolds were then incubated in a FITC-conjugated or TRITC-conjugated secondary antibody (1:200 donkey anti-goat IgG, Santa Cruz Biotechnology) for 45 min in the dark.

    Techniques:

    CMTM7 is localized at the plasma membrane in association with clathrin and sIgM. (A) HeLa cells stably expressing T7-hCMTM7 were fixed, permeabilized, and stained with antibodies for the indicated organelle makers (left panels) and T7 tag (middle panels) as described in the Materials and Methods section. Right panels: merged images. (B) HEK293T cells transiently expressing mouse CMTM7 tagged with HA or FLAG epitopes at the N- or C-termini, respectively, or not (schematically represented at the top), were stained with anti-HA antibody (left) or anti-FLAG antibody (right) and analyzed by flow cytometry. The histograms indicate fluorescence intensities for non-tagged (red lines), and HA- (blue lines) or FLAG- (green lines) tagged CMTM7s. The predicted plasma membrane topology of CMTM7 is depicted at the bottom. (C) BAL17 cells stably expressing T7-tagged mouse CMTM7 (BAL17/T7-CMTM7) were stained with rabbit anti-T7 and biotin-anti-IgM antibodies, and incubated at 37°C for the indicated time periods. Then the cells were fixed, permeabilized, and stained with TRITC-anti-rabbit IgG and FITC-streptavidin. The fluorescence FITC (top), TRITC (middle), and their merged images (bottom) are shown.

    Journal: PLoS ONE

    Article Title: Identification of CMTM7 as a Transmembrane Linker of BLNK and the B-Cell Receptor

    doi: 10.1371/journal.pone.0031829

    Figure Lengend Snippet: CMTM7 is localized at the plasma membrane in association with clathrin and sIgM. (A) HeLa cells stably expressing T7-hCMTM7 were fixed, permeabilized, and stained with antibodies for the indicated organelle makers (left panels) and T7 tag (middle panels) as described in the Materials and Methods section. Right panels: merged images. (B) HEK293T cells transiently expressing mouse CMTM7 tagged with HA or FLAG epitopes at the N- or C-termini, respectively, or not (schematically represented at the top), were stained with anti-HA antibody (left) or anti-FLAG antibody (right) and analyzed by flow cytometry. The histograms indicate fluorescence intensities for non-tagged (red lines), and HA- (blue lines) or FLAG- (green lines) tagged CMTM7s. The predicted plasma membrane topology of CMTM7 is depicted at the bottom. (C) BAL17 cells stably expressing T7-tagged mouse CMTM7 (BAL17/T7-CMTM7) were stained with rabbit anti-T7 and biotin-anti-IgM antibodies, and incubated at 37°C for the indicated time periods. Then the cells were fixed, permeabilized, and stained with TRITC-anti-rabbit IgG and FITC-streptavidin. The fluorescence FITC (top), TRITC (middle), and their merged images (bottom) are shown.

    Article Snippet: Antibodies The following antibodies were used: mouse anti-GM130, HRP-conjugated mouse anti-phosphotyrosine (PY20) (BD Biosciences); goat anti-EEA1 (C-15), goat anti-Btk (C-20), rabbit anti-ERK2 (C-14), rabbit anti-JNK1 (C-17), rabbit anti-Syk (N-19), rabbit anti-PLCγ2 (Q-20), rabbit anti-Lyn (44), FITC-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology); rabbit anti-phospho-p44/42 MAPK (Thr202/Try204), rabbit anti-phospho-PLCγ2 (Tyr1217), rabbit anti-phospho-SAPK/JNK (Tyr183/Tyr185), rabbit anti-phospho-ZAP70/Syk (Tyr319/352), rabbit anti-phospho-Src (Tyr416) (Cell Signaling Technology); mouse anti-calnexin, rabbit anti-T7 (Chemicon); mouse anti-FlagM2 (Sigma-Aldrich); mouse anti-clathrin (Abcam); mouse anti-T7 (Novagen); goat anti-HA (Bethyl Laboratories); goat F(ab′)2 anti-mouse IgM, TRITC-conjugated donkey anti-rabbit IgG, HRP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch); biotin-goat F(ab′)2 anti-mouse IgM, FITC-conjugated goat anti-mouse IgG, HRP-conjugated goat anti-mouse IgM (Southern Biotech); goat anti-mouse IgM (Cappel); HRP-conjugated rabbit anti-mouse IgG (Zymed); FITC-conjugated streptavidin (Biolegend); rabbit anti-BLNK (Hayashi et al., 2000).

    Techniques: Stable Transfection, Expressing, Staining, Flow Cytometry, Cytometry, Fluorescence, Incubation

    Integrin VLA-3 is a receptor for APC (A) Binding assay of soluble VLA-3, VLA-5, and α V β 3 to APC. Soluble integrins were incubated with the indicated amount of rhAPC in the presence of 1 mM MnCl 2 + integrin activating Abs. The protein complexes were precipitated and subjected to SDS-PAGE and western blotting with an APC Ab (upper panel). The total amounts of soluble integrins precipitated were shown by silver staining (lower panels). (B) rhAPC was incubated with human neutrophils in the presence of 1 mM MnCl 2 . The cells were pretreated with control IgG (Con IgG) or blocking antibodies against integrins VLA-3 (α 3 ), VLA-5 (α 5 ), α V β 3 (β 3 ), and Mac-1 (α M ). Bound APC was detected by flow cytometry. APC binding in the presence of integrin blocking antibodies is expressed as the percentage increase in MFI, compared with the cells not treated with APC (control) in the presence of the same blocking antibody. The data shows the mean ± SEM of three donors. * indicates significance between Con IgG vs. anti-VLA-3 (α 3 ). * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Activated protein C attenuates severe inflammation by targeting VLA-3high neutrophil subpopulation in mice

    doi: 10.4049/jimmunol.1700541

    Figure Lengend Snippet: Integrin VLA-3 is a receptor for APC (A) Binding assay of soluble VLA-3, VLA-5, and α V β 3 to APC. Soluble integrins were incubated with the indicated amount of rhAPC in the presence of 1 mM MnCl 2 + integrin activating Abs. The protein complexes were precipitated and subjected to SDS-PAGE and western blotting with an APC Ab (upper panel). The total amounts of soluble integrins precipitated were shown by silver staining (lower panels). (B) rhAPC was incubated with human neutrophils in the presence of 1 mM MnCl 2 . The cells were pretreated with control IgG (Con IgG) or blocking antibodies against integrins VLA-3 (α 3 ), VLA-5 (α 5 ), α V β 3 (β 3 ), and Mac-1 (α M ). Bound APC was detected by flow cytometry. APC binding in the presence of integrin blocking antibodies is expressed as the percentage increase in MFI, compared with the cells not treated with APC (control) in the presence of the same blocking antibody. The data shows the mean ± SEM of three donors. * indicates significance between Con IgG vs. anti-VLA-3 (α 3 ). * p

    Article Snippet: Allophycocyanin (APC) labeled Ly6G (BD Biosciences, San Diego, CA), PerCp-Cy5.5 labeled anti-CD11b (eBioscience, San Diego, CA,), fluorescein isothiocyanate (FITC) anti-F4/80 (eBioscience, San Diego, CA, USA), purified goat anti-mouse integrin α3 /CD49c (R & D Systems), and PE conjugated donkey anti-goat IgG (Santa Cruz) antibodies were used.

    Techniques: Binding Assay, Incubation, SDS Page, Western Blot, Silver Staining, Blocking Assay, Flow Cytometry, Cytometry

    p53 represses the K14 promoter indirectly by associating with transcription factor SP1. (A) The sequence map of the first 160 base pairs of the K14 promoter. No p53 consensus binding site was found within the K14 promoter based on a search for the following typical p53 consensus binding site: PuPuPuC(T/A)(T/A)GPyPyPy-N(013)-PuPuPuC(T/A)(T/A)GpyPyPy; N(0–13) indicates a spacer of 0 to 13 bases. There is an SP1 binding site between base pairs -48 and -43 and a TATA box binding site located between base pairs -29 and -24. The K14 promoter sequence from the GenBank database [GenBank: HSU11076] is shown, and the transcription initiation site refers to the NCBI reference sequence of the human K14 gene [GenBank: NM_000526.4]. (B) H1299 cells were co-transfected with wild-type K14 (pK14WT-160), the TATA site mutant (pK14mtTATA-160), or the SP1 site mutant (pK14mtSP1-160) promoter vectors and pcDNA 3.0 or wild-type p53 expression vectors. The levels of luciferase signal were normalized to the luciferase level of pcDNA 3.0, which was set to 1. p53 was able to repress luciferase expression driven by the pK14WT-160 and pK14mtTATA-160 promoter constructs, but not the pK14mtSP1-160 promoter construct. (C) H1299 cells were co-transfected with pK14-2000 or pK14-160 promoter constructs and pcDNA 3.0, wild-type p53, or p53Δ293-393 expression vectors. The level of luciferase signal from each sample was normalized to the pcDNA 3.0 sample, which was set to 1. p53 but not p53Δ293-393 suppressed luciferase expression driven by the pK14-2000 and pK14-160 promoters. (D) DAPA was used to purify the nuclear protein extract from H1299 cells transfected with the p53Δ363-393 or p53Δ293-393 expression vector. Nuclear proteins were incubated with oligonucleotide probes corresponding to base pairs -53 to -38 of the K14 promoter with a wild-type SP1 binding site CCCGCC or with an SP1 binding site mutant GGTACC. Nuclear proteins that co-precipitated with the probes were blotted with anti-SP1 or anti-p53 antibodies. The SP1 binding site on the K14 promoter co-precipitated with SP1 and p53Δ363-393 but not p53Δ293-393. The SP1 binding site mutant probe was unable to pull down SP1, p53Δ363-393, or p53Δ293-393. “5% input” denotes the positive loading control for nuclear protein, and “beads only” denotes the non-probed negative control. (E) In the ChIP assay, Sp1 and p53 antibodies immunoprecipitated base pair -220 to -5 of the K14 promoter in C9 cells. Non-specific IgG antibody was included as the negative control. IgG, Sp1 or p53 antibody was unable to immunoprecipitate the K14 promoter fragment from base pair -689 to -551. (I: input; G: IgG).

    Journal: PLoS ONE

    Article Title: p53 Acts as a Co-Repressor to Regulate Keratin 14 Expression during Epidermal Cell Differentiation

    doi: 10.1371/journal.pone.0041742

    Figure Lengend Snippet: p53 represses the K14 promoter indirectly by associating with transcription factor SP1. (A) The sequence map of the first 160 base pairs of the K14 promoter. No p53 consensus binding site was found within the K14 promoter based on a search for the following typical p53 consensus binding site: PuPuPuC(T/A)(T/A)GPyPyPy-N(013)-PuPuPuC(T/A)(T/A)GpyPyPy; N(0–13) indicates a spacer of 0 to 13 bases. There is an SP1 binding site between base pairs -48 and -43 and a TATA box binding site located between base pairs -29 and -24. The K14 promoter sequence from the GenBank database [GenBank: HSU11076] is shown, and the transcription initiation site refers to the NCBI reference sequence of the human K14 gene [GenBank: NM_000526.4]. (B) H1299 cells were co-transfected with wild-type K14 (pK14WT-160), the TATA site mutant (pK14mtTATA-160), or the SP1 site mutant (pK14mtSP1-160) promoter vectors and pcDNA 3.0 or wild-type p53 expression vectors. The levels of luciferase signal were normalized to the luciferase level of pcDNA 3.0, which was set to 1. p53 was able to repress luciferase expression driven by the pK14WT-160 and pK14mtTATA-160 promoter constructs, but not the pK14mtSP1-160 promoter construct. (C) H1299 cells were co-transfected with pK14-2000 or pK14-160 promoter constructs and pcDNA 3.0, wild-type p53, or p53Δ293-393 expression vectors. The level of luciferase signal from each sample was normalized to the pcDNA 3.0 sample, which was set to 1. p53 but not p53Δ293-393 suppressed luciferase expression driven by the pK14-2000 and pK14-160 promoters. (D) DAPA was used to purify the nuclear protein extract from H1299 cells transfected with the p53Δ363-393 or p53Δ293-393 expression vector. Nuclear proteins were incubated with oligonucleotide probes corresponding to base pairs -53 to -38 of the K14 promoter with a wild-type SP1 binding site CCCGCC or with an SP1 binding site mutant GGTACC. Nuclear proteins that co-precipitated with the probes were blotted with anti-SP1 or anti-p53 antibodies. The SP1 binding site on the K14 promoter co-precipitated with SP1 and p53Δ363-393 but not p53Δ293-393. The SP1 binding site mutant probe was unable to pull down SP1, p53Δ363-393, or p53Δ293-393. “5% input” denotes the positive loading control for nuclear protein, and “beads only” denotes the non-probed negative control. (E) In the ChIP assay, Sp1 and p53 antibodies immunoprecipitated base pair -220 to -5 of the K14 promoter in C9 cells. Non-specific IgG antibody was included as the negative control. IgG, Sp1 or p53 antibody was unable to immunoprecipitate the K14 promoter fragment from base pair -689 to -551. (I: input; G: IgG).

    Article Snippet: Goat anti-mouse, goat anti-rabbit, or donkey anti-goat immunoglobulin G (IgG) conjugated with horseradish peroxidase was used as the secondary antibody (Santa Cruz).

    Techniques: Sequencing, Binding Assay, Transfection, Mutagenesis, Expressing, Luciferase, Construct, Plasmid Preparation, Incubation, Negative Control, Chromatin Immunoprecipitation, Immunoprecipitation