Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Localization of Hsp90, Hsp70, Hsp60, Hsp40, PDI and integrin β3 in cell membrane. A. Cells were fixed in 4% PFD and incubated with goat primary antibodies against the above-mentioned proteins. Alexa Fluor488-conjugate donkey anti-goat IgG was used as secondary antibody. Immunofluorescence analysis was performed in duplicate using a Cyan (Dako) flow cytometer and a FlowJo software. B-D. RIPA buffer-solubilized cell membrane-enriched fractions were separately added to ELISA plates coated with rabbit capture antibodies against Hsc70, PDI or integrin β3. Goat primary antibodies to Hsp90, Hsp70, Hsp60, Hsp40 Hsc70, PDI or integrin β3 were added for detection before addition of secondary HRP-conjugated donkey anti-goat antibody. Plates were read at 492 nm and the results expressed as delta OD after subtracting control values. E, F. Cells with (E) or without (F) methyl- β-cyclodextrin treatment were lysed and subjected to sucrose gradient centrifugation before collection of gradient fractions containing rafts. The RIPA buffer-solubilized fractions were added to ELISA plates coated with rabbit antibodies to integrin β3. Goat primary antibodies to Hsp90, Hsp70, Hsp60, Hsp40 or integrin β3 were added and then secondary HRP-conjugated donkey anti-goat IgG. The reaction was visualized using OPD and the plates read at 492 nm. Data are shown as means ( standard deviation from three independent experiments performed in duplicate.

Article Snippet: The casein-blocked membranes were incubated with a mix of goat antibodies (0.2 μg/mL each, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 and Hsp40 or alternatively with rabbit antibodies against rotavirus SP for 1 h at 37° C. In the case of MA104 cells with Wt1-5, antibodies against integrin β3, Hsc70, PDI or HSPs (90, 70, 60, 40) were used in the SDS-PAGE/Western blotting analysis.

Techniques: Membrane, Incubation, Immunofluorescence, Flow Cytometry, Software, Enzyme-linked Immunosorbent Assay, Control, Gradient Centrifugation, Standard Deviation

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Binding of rotavirus isolates to Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, PDI and integrin β3. MCF-7 cells (A-E) or their cell membrane enriched fractions (F-J) were incubated with the indicated rotavirus isolates. After PBS-washing, the preparations were RIPA-solubilized and applied into ELISA plates coated with rabbit capture antibodies to rotavirus structural proteins. For detection, goat antibodies to HSPs, PDI, Hsc70, integrin β3 or to rotavirus were used and the reaction measured using secondary HRP-conjugated donkey anti-goat IgG and OPD substrate.

Article Snippet: The casein-blocked membranes were incubated with a mix of goat antibodies (0.2 μg/mL each, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 and Hsp40 or alternatively with rabbit antibodies against rotavirus SP for 1 h at 37° C. In the case of MA104 cells with Wt1-5, antibodies against integrin β3, Hsc70, PDI or HSPs (90, 70, 60, 40) were used in the SDS-PAGE/Western blotting analysis.

Techniques: Binding Assay, Membrane, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Binging of rotavirus particles to Hsp90, Hsp70, Hsp60 and Hsp40, and inhibition of rotavirus infection by competition for binding to these HSPs. A. MCF-7 cell membrane-enriched fractions were separately incubated with the indicated rotavirus isolates. The solubilized cell membrane fractions were immunoprecipitated with agarose bead-coupled antibodies against rotavirus particles. The immunoprecipitated proteins were analyzed by SDS-PAGE/Western blotting using antibodies against the indicated HSPs and luminol-based detection. B. MA104 cell membrane-enriched fractions were incubated with rotavirus isolate. The solubilized cell membrane fractions were immunoprecipitated with agarose bead-coupled antibodies against rotavirus particles. The immunoprecipitated proteins were analyzed by SDS-PAGE/Western blotting using antibodies against integrin β3, Hsc70, and PDI or HSPs and luminol-based detection. C. A VOBPA assay was conducted by lysing MCF-7 cells with RIPA buffer followed by non-denaturing and non-reducing PAGE and Western blotting. The membrane was incubated with rotavirus WTEW, washed quickly and treated with 2.5% glutaraldehyde. The membrane was treated with a mix of antibodies to the HSPs (lane 1) indicated above or antibodies to rotavirus TLPs (lane 2). Reactions were revealed with Western blotting Luminol Reagent. D, E. Rotavirus isolates (TRUYO, WWM, WTEW, WT-15 and ECwt-o) at MOI 0.8 were treated with the indicated concentrations of rHsp90 (D) or rHsp70 (E) before incubation with MCF-cells. Rotavirus isolates were treated with BSA (100 ng/mL) or Hsc70 (100 ng/mL) or left untreated to be used as controls. The frozen and thawed cells were treated with RIPA buffer and the lysate applied on ELISA plates coated with Guinea pig capture antibodies to rotavirus particles. Detection was performed with rabbit primary antibodies to rotavirus particles. The reaction was revealed with secondary HRP-conjugated goat anti-rabbit IgG and OPD. F. Cell viability of cells treated with recombinant proteins (rHsp90 or rHsp70) (100 (g/mL) was determined using trypan blue exclusion test. Data are presented as means ( standard deviation from three independent experiments performed in duplicate.

Article Snippet: The casein-blocked membranes were incubated with a mix of goat antibodies (0.2 μg/mL each, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 and Hsp40 or alternatively with rabbit antibodies against rotavirus SP for 1 h at 37° C. In the case of MA104 cells with Wt1-5, antibodies against integrin β3, Hsc70, PDI or HSPs (90, 70, 60, 40) were used in the SDS-PAGE/Western blotting analysis.

Techniques: Inhibition, Infection, Binding Assay, Membrane, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Localización de Hsp90, Hsp70, Hsp60, Hsp40, PDI e integrina β3 en la membrana celular. A. Las células se fijaron en PFD al 4% y se incubaron con anticuerpos primarios de cabra contra las proteínas mencionadas anteriormente. Se usó como anticuerpo secundario IgG de burro anti-cabra conjugado con Alexa Fluor 488. El análisis de inmunofluorescencia se realizó por duplicado utilizando un citómetro de flujo Cyan (Dako) y un software FlowJo. B-D. Se añadieron por separado fracciones enriquecidas en membranas celulares solubilizadas en tampón RIPA a placas de ELISA recubiertas con anticuerpos de captura de conejo contra Hsc70, PDI o integrina β3. Se añadieron anticuerpos primarios de cabra contra Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, PDI o integrina β3 para la detección. Luego se adicionó anticuerpo secundario de burro anti-cabra conjugado con HRP. Las placas se leyeron a 492 nm y los resultados se expresaron como delta OD después de restar los valores del control. E, F. Las células con (E) o sin (F) tratamiento con metil-β-ciclodextrina se lisaron y se sometieron a centrifugación en gradiente de sacarosa. Luego, se recolectaron las fracciones del gradiente que contenían las balsas lipídicas ( rafts ). Las fracciones se solubilizaron en tampón de RIPA y se añadieron a placas de ELISA recubiertas con anticuerpos de conejo contra la integrina β3. Se añadieron anticuerpos primarios de cabra contra Hsp90, Hsp70, Hsp60, Hsp40 o integrina β3 y luego anticuerpo secundario de burro IgG anti-cabra conjugado con HRP. La reacción se visualizó usando OPD y las placas se leyeron a 492 nm. Los datos se muestran como promedio ± desviación estándar (DS) de tres experimentos independientes realizados por duplicado.

Article Snippet: The casein-blocked membranes were incubated with a mix of goat antibodies (0.2 μg/mL each, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 and Hsp40 or alternatively with rabbit antibodies against rotavirus SP for 1 h at 37° C. In the case of MA104 cells with Wt1-5, antibodies against integrin β3, Hsc70, PDI or HSPs (90, 70, 60, 40) were used in the SDS-PAGE/Western blotting analysis.

Techniques: Software, Enzyme-linked Immunosorbent Assay, Control

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Unión de aislamientos de rotavirus a Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, PDI e integrina β3. Las células MCF-7 (A-E) o sus fracciones enriquecidas de membrana celular (F-J) se incubaron con los aislamientos de rotavirus indicados. Después del lavado con PBS, las preparaciones se solubilizaron con RIPA y se aplicaron en placas de ELISA recubiertas con anticuerpos de captura de conejo contra proteínas estructurales de rotavirus. Para la detección, se usaron anticuerpos de cabra contra HSP, PDI, Hsc70, integrina β3 o contra rotavirus y se midió la reacción usando anticuerpo secundario de burro anti-IgG de cabra conjugado con HRP y se reveló usando sustrato de OPD.

Article Snippet: The casein-blocked membranes were incubated with a mix of goat antibodies (0.2 μg/mL each, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 and Hsp40 or alternatively with rabbit antibodies against rotavirus SP for 1 h at 37° C. In the case of MA104 cells with Wt1-5, antibodies against integrin β3, Hsc70, PDI or HSPs (90, 70, 60, 40) were used in the SDS-PAGE/Western blotting analysis.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Unión de partículas de rotavirus a Hsp90, Hsp70, Hsp60 y Hsp40, e inhibición de la infección por rotavirus al competir con HSPs de la célula. A. Las fracciones enriquecidas en la membrana celular de MCF-7 se incubaron por separado con los aislados de rotavirus indicados. Las fracciones de membrana celular solubilizadas se inmunoprecipitaron con anticuerpos acoplados a perlas de agarosa contra partículas de rotavirus. Las proteínas inmunoprecipitadas se analizaron mediante SDS-PAGE/Western blotting usando anticuerpos contra las HSPs indicadas y detección basada en luminol. B. Se incubaron fracciones enriquecidas en la membrana celular MA104 con aislado de rotavirus. Las fracciones de membrana celular solubilizadas se inmunoprecipitaron con anticuerpos acoplados a perlas de agarosa contra partículas de rotavirus. Las proteínas inmunoprecipitadas se analizaron mediante SDS-PAGE/Western blotting usando anticuerpos contra la integrina β3, Hsc70, PDI o HSP y detección basada en luminol. C. Se realizó un ensayo de VOBPA lisando células MCF-7 con tampón RIPA seguido de PAGE no desnaturalizante y no reductor y transferencia Western. La membrana se incubó con rotavirus WTEW, se lavó rápidamente y se trató con glutaraldehído al 2,5%. La membrana se trató con una mezcla de anticuerpos contra las HSPs (carril 1) indicadas anteriormente o anticuerpos contra las TLPs de rotavirus (carril 2). Las reacciones se revelaron con reactivo Western Luminol de transferencia. D, E. Los aislamientos de rotavirus (TRUYO, WWM, WTEW, WT-15 y ECwt-o) a un MOI 0.8 se trataron con las concentraciones indicadas de rHsp90 (D) o rHsp70 (E) antes de la incubación con células MCF-7. Los aislamientos de rotavirus se trataron con BSA (100 ng/mL) o Hsc70 (100 ng/ml) o se dejaron sin tratar para utilizarlos como controles. Las células congeladas y descongeladas se trataron con tampón RIPA y el lisado se aplicó sobre placas ELISA recubiertas con anticuerpos de captura de cobayo contra partículas de rotavirus. La detección se realizó con anticuerpos primarios de conejo contra partículas de rotavirus. La reacción se reveló con anticuerpos secundarios IgG de cabra anti-conejo conjugados con HRP y se revelo con sustrato de OPD. F. Se determinó la viabilidad celular de las células tratadas con proteínas recombinantes (rHsp90 o rHsp70) (100 µg/mL) usando una prueba de exclusión con azul de tripano. Los datos se presentan como promedios ± desviación estándar (DS) de tres experimentos independientes realizados por duplicado.

Article Snippet: The casein-blocked membranes were incubated with a mix of goat antibodies (0.2 μg/mL each, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 and Hsp40 or alternatively with rabbit antibodies against rotavirus SP for 1 h at 37° C. In the case of MA104 cells with Wt1-5, antibodies against integrin β3, Hsc70, PDI or HSPs (90, 70, 60, 40) were used in the SDS-PAGE/Western blotting analysis.

Techniques: SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet:

Article Snippet: The casein-blocked membranes were incubated with a mix of goat antibodies (0.2 μg/mL each, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 and Hsp40 or alternatively with rabbit antibodies against rotavirus SP for 1 h at 37° C. In the case of MA104 cells with Wt1-5, antibodies against integrin β3, Hsc70, PDI or HSPs (90, 70, 60, 40) were used in the SDS-PAGE/Western blotting analysis.

Techniques: Derivative Assay, In Vitro, Cell Surface Receptor Assay

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet:

Article Snippet: The casein-blocked membranes were incubated with a mix of goat antibodies (0.2 μg/mL each, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 and Hsp40 or alternatively with rabbit antibodies against rotavirus SP for 1 h at 37° C. In the case of MA104 cells with Wt1-5, antibodies against integrin β3, Hsc70, PDI or HSPs (90, 70, 60, 40) were used in the SDS-PAGE/Western blotting analysis.

Techniques: In Vitro, Virus

Journal: Arthritis Research & Therapy

Article Title: Effect of Porphyromonas gingivalis infection on gut dysbiosis and resultant arthritis exacerbation in mouse model

doi: 10.1186/s13075-020-02348-z

Figure Lengend Snippet: Effect of PgPAD on the onset of joint arthritis. The effect of PgPAD on the onset of arthritis was determined. In this experiment, PgPAD deletion mutant 33277 strain (⊿ pad ) and Pg 33277 wild-type (WT) strain were used, unless Pg W83 strain was specifically described in caption of Figures. ABL of the upper jaw in each group was measured ( a ). Ctrl, PBS inoculation; WT, Pg wild-type inoculation; ⊿ pad , Pg pad knockout strain inoculation. To evaluate inflammation of the gingival tissue 6 weeks after Pg inoculation, IL-6 production in gingival tissue of SKG mice was measured by ELISA ( b ). AS was measured 3 and 6 weeks after Pg inoculation with LA i.p. injection ( c ). To assess CP synthesis, ELISA was performed ( d gingival tissue, e joint tissue, f small intestine, g large intestine). The titer of serum ACPA 6 weeks after Pg inoculation with LA i.p. injection in mice from each group was measured by ELISA ( h ). The production of endogenous PADI2 and PADI4 in joint tissue was determined by Western blotting, and then the density of each band was measured by NIH Image-J ( i and j ). Data represent mean ± SD ( b – j ) of 5 mice per group. Statistical analyses were performed using the Tukey-Kramer test and Bonferroni-corrected Mann-Whitney U test for multiple comparisons (* P < 0.05, ** P < 0.01)

Article Snippet: As a control, the amount of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected by anti-GAPDH antibody (10 μg/ml, HRP-60004, PROTEINTECH JAPAN, Japan) The density of target PADI2 and PADI4 bands was measured by NIH Image-J software.

Techniques: Mutagenesis, Knock-Out, Enzyme-linked Immunosorbent Assay, Injection, Western Blot, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Effect of Porphyromonas gingivalis infection on gut dysbiosis and resultant arthritis exacerbation in mouse model

doi: 10.1186/s13075-020-02348-z

Figure Lengend Snippet: Effect of PgPAD on the onset of joint arthritis. The effect of PgPAD on the onset of arthritis was determined. In this experiment, PgPAD deletion mutant 33277 strain (⊿ pad ) and Pg 33277 wild-type (WT) strain were used, unless Pg W83 strain was specifically described in caption of Figures. ABL of the upper jaw in each group was measured ( a ). Ctrl, PBS inoculation; WT, Pg wild-type inoculation; ⊿ pad , Pg pad knockout strain inoculation. To evaluate inflammation of the gingival tissue 6 weeks after Pg inoculation, IL-6 production in gingival tissue of SKG mice was measured by ELISA ( b ). AS was measured 3 and 6 weeks after Pg inoculation with LA i.p. injection ( c ). To assess CP synthesis, ELISA was performed ( d gingival tissue, e joint tissue, f small intestine, g large intestine). The titer of serum ACPA 6 weeks after Pg inoculation with LA i.p. injection in mice from each group was measured by ELISA ( h ). The production of endogenous PADI2 and PADI4 in joint tissue was determined by Western blotting, and then the density of each band was measured by NIH Image-J ( i and j ). Data represent mean ± SD ( b – j ) of 5 mice per group. Statistical analyses were performed using the Tukey-Kramer test and Bonferroni-corrected Mann-Whitney U test for multiple comparisons (* P < 0.05, ** P < 0.01)

Article Snippet: As a control, the amount of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected by anti-GAPDH antibody (10 μg/ml, HRP-60004, PROTEINTECH JAPAN, Japan) The density of target PADI2 and PADI4 bands was measured by NIH Image-J software.

Techniques: Mutagenesis, Knock-Out, Enzyme-linked Immunosorbent Assay, Injection, Western Blot, MANN-WHITNEY

Journal: iScience

Article Title: Advanced oxidation protein products induce Paneth cells defects by endoplasmic reticulum stress in Crohn's disease

doi: 10.1016/j.isci.2023.107312

Figure Lengend Snippet:

Article Snippet: Rabbit PDI , Proteintech , Cat#11245-1-AP.

Techniques: Recombinant, Lysis, Protein Quantitation, Isolation, ATP Assay, Software

Journal: Cell reports

Article Title: Activation of endoplasmic reticulum stress in premature aging via the inner nuclear membrane protein SUN2

doi: 10.1016/j.celrep.2023.112534

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-PDI, Clone RL90 , Abcam , Cat# ab2792; RRID:AB_303304.

Techniques: Produced, Marker, Recombinant, SYBR Green Assay, Transgenic Assay, Expressing, Knock-In, Negative Control, Real-time Polymerase Chain Reaction, Derivative Assay, Plasmid Preparation, Construct, Software, Imaging, Light Microscopy, Laser-Scanning Microscopy, Microscopy, FACS

Journal: BMC Cancer

Article Title: BB-Cl-Amidine as a novel therapeutic for canine and feline mammary cancer via activation of the endoplasmic reticulum stress pathway

doi: 10.1186/s12885-018-4323-8

Figure Lengend Snippet: Primers used in the study

Article Snippet: The rabbit anti-human PAD2 and PAD4 antibodies were obtained from Proteintech (12110–1-AP; Rosemont, IL) and Sigma-Aldrich (P4749; St. Louis, MO), respectively.

Techniques:

Journal: BMC Cancer

Article Title: BB-Cl-Amidine as a novel therapeutic for canine and feline mammary cancer via activation of the endoplasmic reticulum stress pathway

doi: 10.1186/s12885-018-4323-8

Figure Lengend Snippet: Expression of PAD2 and PAD4 in non-malignant and tumoral canine and feline mammary cell lines. a Expression levels of the genes PAD2 and PAD4 in canine and feline normal and tumoral mammary cell lines as determined by qRT-PCR. b Protein expression in whole cell lysates subjected to SDS-PAGE and immunoblot analyses probed with PAD2 or PAD4 antibodies. β-actin was included as a loading control. Representative Western blots and quantifications are shown. Quantification is represented as the fold change of PAD2/4 band density over β-actin band density. c Protein expression in cells probed with either PAD2 or PAD4 antibodies and subjected to flow cytometry analyses. d Protein expression in whole cell lysates subjected to SDS-PAGE and immunoblot analyses probed with anti-modified citrulline antibodies. β-actin was included as a loading control. Representative Western blots and quantifications are shown. Quantification is represented as the fold change of modified-citrulline band density over β-actin band density. * P < 0.05, n = 3. Data are presented as mean ± standard deviation

Article Snippet: The rabbit anti-human PAD2 and PAD4 antibodies were obtained from Proteintech (12110–1-AP; Rosemont, IL) and Sigma-Aldrich (P4749; St. Louis, MO), respectively.

Techniques: Expressing, Quantitative RT-PCR, SDS Page, Western Blot, Control, Flow Cytometry, Modification, Standard Deviation

Journal: BMC Cancer

Article Title: BB-Cl-Amidine as a novel therapeutic for canine and feline mammary cancer via activation of the endoplasmic reticulum stress pathway

doi: 10.1186/s12885-018-4323-8

Figure Lengend Snippet: Cellular localization of PAD2 and PAD4 in non-malignant and tumoral canine and feline mammary cell lines. Protein localization in cells probed with PAD2 or PAD4 antibodies and subjected to immunofluorescence. Representative fluorescence pictures ( a ) and quantifications, using Image J Software, ( b ) are shown. ** P < 0.01, n = 3. Data are presented as mean ± standard deviation

Article Snippet: The rabbit anti-human PAD2 and PAD4 antibodies were obtained from Proteintech (12110–1-AP; Rosemont, IL) and Sigma-Aldrich (P4749; St. Louis, MO), respectively.

Techniques: Immunofluorescence, Fluorescence, Software, Standard Deviation