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: After centrifugation at 120,000 g for 4 h at 4° C, fractions (500 μL) were collected from the top of the gradient and the fractions containing the rafts were treated with 1% Triton X-100 for 30 min at 37° C. The solubilized fractions were applied to ELISA plates coated with rabbit polyclonal antibodies against integrin β3, blocked with 0.6% casein and then incubated overnight at 4° C. After washing three times with PBS-T, the plate wells were treated with primary goat antibodies (0.2 μg/mL, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 or Hsp40 for 1 h at 37o C. Following three times washing with PBS-T, secondary HRP-conjugated donkey anti-goat IgG (0.133 μg/mL, Santa Cruz Biotechnology Inc. SC-2020) was added, followed by incubation for 1 h at 37o C. After PBS-T washing three times, OPD was used to visualize and quantify the reaction as indicated above.

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: After centrifugation at 120,000 g for 4 h at 4° C, fractions (500 μL) were collected from the top of the gradient and the fractions containing the rafts were treated with 1% Triton X-100 for 30 min at 37° C. The solubilized fractions were applied to ELISA plates coated with rabbit polyclonal antibodies against integrin β3, blocked with 0.6% casein and then incubated overnight at 4° C. After washing three times with PBS-T, the plate wells were treated with primary goat antibodies (0.2 μg/mL, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 or Hsp40 for 1 h at 37o C. Following three times washing with PBS-T, secondary HRP-conjugated donkey anti-goat IgG (0.133 μg/mL, Santa Cruz Biotechnology Inc. SC-2020) was added, followed by incubation for 1 h at 37o C. After PBS-T washing three times, OPD was used to visualize and quantify the reaction as indicated above.

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: Blocking of rotavirus binding to cell surface by antibodies against Hsp90, Hsp70, Hsp60, Hsp40, and Hsc70. A-F. MCF-7 cells were incubated with F(ab’)2 antibody fragments against to Hsp90, Hsp70, Hsc70, Hsp60 or Hsp40 at the indicated dilutions, followed by incubation with the rotavirus isolates indicated in panels. After PBS- washing, the cells were solubilized with RIPA buffer before addition to ELISA wells coated with guinea pig capture antibodies to rotavirus particles. For detection, rabbit antibodies to rotavirus particles were used and the reaction measured using secondary HRP-conjugated donkey anti-rabbit IgG and OPD substrate. Data are means values ( standard deviation from three independent experiments performed in duplicate.

Article Snippet: After centrifugation at 120,000 g for 4 h at 4° C, fractions (500 μL) were collected from the top of the gradient and the fractions containing the rafts were treated with 1% Triton X-100 for 30 min at 37° C. The solubilized fractions were applied to ELISA plates coated with rabbit polyclonal antibodies against integrin β3, blocked with 0.6% casein and then incubated overnight at 4° C. After washing three times with PBS-T, the plate wells were treated with primary goat antibodies (0.2 μg/mL, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 or Hsp40 for 1 h at 37o C. Following three times washing with PBS-T, secondary HRP-conjugated donkey anti-goat IgG (0.133 μg/mL, Santa Cruz Biotechnology Inc. SC-2020) was added, followed by incubation for 1 h at 37o C. After PBS-T washing three times, OPD was used to visualize and quantify the reaction as indicated above.

Techniques: Blocking Assay, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, 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: 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: After centrifugation at 120,000 g for 4 h at 4° C, fractions (500 μL) were collected from the top of the gradient and the fractions containing the rafts were treated with 1% Triton X-100 for 30 min at 37° C. The solubilized fractions were applied to ELISA plates coated with rabbit polyclonal antibodies against integrin β3, blocked with 0.6% casein and then incubated overnight at 4° C. After washing three times with PBS-T, the plate wells were treated with primary goat antibodies (0.2 μg/mL, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 or Hsp40 for 1 h at 37o C. Following three times washing with PBS-T, secondary HRP-conjugated donkey anti-goat IgG (0.133 μg/mL, Santa Cruz Biotechnology Inc. SC-2020) was added, followed by incubation for 1 h at 37o C. After PBS-T washing three times, OPD was used to visualize and quantify the reaction as indicated above.

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: Decrease of rotavirus infection by antibodies to Hsp90, Hsp70, Hsp60 or Hsp40. A-E. MCF-7 cells in culture medium were separately treated with a serial dilution of antibodies to the indicated HSPs for 45 min at 4° C, DMEM-washed and then incubated with the rotavirus isolates indicated in panels. After 12 h.p.i., structural rotavirus antigens were detected by immunocytochemistry assay. Virus inoculated cells without antibody pre-treatment were used as a control. Data were expressed as percentage of infected cells. Data are shown as mean percentages ( standard deviation from three independent experiments performed in duplicate.

Article Snippet: After centrifugation at 120,000 g for 4 h at 4° C, fractions (500 μL) were collected from the top of the gradient and the fractions containing the rafts were treated with 1% Triton X-100 for 30 min at 37° C. The solubilized fractions were applied to ELISA plates coated with rabbit polyclonal antibodies against integrin β3, blocked with 0.6% casein and then incubated overnight at 4° C. After washing three times with PBS-T, the plate wells were treated with primary goat antibodies (0.2 μg/mL, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 or Hsp40 for 1 h at 37o C. Following three times washing with PBS-T, secondary HRP-conjugated donkey anti-goat IgG (0.133 μg/mL, Santa Cruz Biotechnology Inc. SC-2020) was added, followed by incubation for 1 h at 37o C. After PBS-T washing three times, OPD was used to visualize and quantify the reaction as indicated above.

Techniques: Infection, Serial Dilution, Incubation, Immunocytochemistry, Virus, Control, 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: After centrifugation at 120,000 g for 4 h at 4° C, fractions (500 μL) were collected from the top of the gradient and the fractions containing the rafts were treated with 1% Triton X-100 for 30 min at 37° C. The solubilized fractions were applied to ELISA plates coated with rabbit polyclonal antibodies against integrin β3, blocked with 0.6% casein and then incubated overnight at 4° C. After washing three times with PBS-T, the plate wells were treated with primary goat antibodies (0.2 μg/mL, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 or Hsp40 for 1 h at 37o C. Following three times washing with PBS-T, secondary HRP-conjugated donkey anti-goat IgG (0.133 μg/mL, Santa Cruz Biotechnology Inc. SC-2020) was added, followed by incubation for 1 h at 37o C. After PBS-T washing three times, OPD was used to visualize and quantify the reaction as indicated above.

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: After centrifugation at 120,000 g for 4 h at 4° C, fractions (500 μL) were collected from the top of the gradient and the fractions containing the rafts were treated with 1% Triton X-100 for 30 min at 37° C. The solubilized fractions were applied to ELISA plates coated with rabbit polyclonal antibodies against integrin β3, blocked with 0.6% casein and then incubated overnight at 4° C. After washing three times with PBS-T, the plate wells were treated with primary goat antibodies (0.2 μg/mL, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 or Hsp40 for 1 h at 37o C. Following three times washing with PBS-T, secondary HRP-conjugated donkey anti-goat IgG (0.133 μg/mL, Santa Cruz Biotechnology Inc. SC-2020) was added, followed by incubation for 1 h at 37o C. After PBS-T washing three times, OPD was used to visualize and quantify the reaction as indicated above.

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: Bloqueo de la unión de rotavirus a la superficie celular con anticuerpos contra Hsp90, Hsp70, Hsp60, Hsp40 y Hsc70. A-F. Las células MCF-7 se incubaron con fragmentos de anticuerpo F(ab')2 contra Hsp90, Hsp70, Hsc70, Hsp60 o Hsp40 en las diluciones indicadas, seguido de incubación con los aislados de rotavirus indicados en los paneles. Después del lavado con PBS, las células se solubilizaron con tampón RIPA antes de la adición a los pozos de ELISA recubiertos con anticuerpos de captura de cobayo contra proteínas estructurales de rotavirus. Para la detección, se usaron anticuerpos de conejo contra partículas de rotavirus y se midió la reacción usando IgG secundario de burro anti-conejo conjugado con HRP y se reveló utilizando sustrato de OPD. Los datos son valores medios ± desviación estándar de tres experimentos independientes realizados por duplicado.

Article Snippet: After centrifugation at 120,000 g for 4 h at 4° C, fractions (500 μL) were collected from the top of the gradient and the fractions containing the rafts were treated with 1% Triton X-100 for 30 min at 37° C. The solubilized fractions were applied to ELISA plates coated with rabbit polyclonal antibodies against integrin β3, blocked with 0.6% casein and then incubated overnight at 4° C. After washing three times with PBS-T, the plate wells were treated with primary goat antibodies (0.2 μg/mL, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 or Hsp40 for 1 h at 37o C. Following three times washing with PBS-T, secondary HRP-conjugated donkey anti-goat IgG (0.133 μg/mL, Santa Cruz Biotechnology Inc. SC-2020) was added, followed by incubation for 1 h at 37o C. After PBS-T washing three times, OPD was used to visualize and quantify the reaction as indicated above.

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: After centrifugation at 120,000 g for 4 h at 4° C, fractions (500 μL) were collected from the top of the gradient and the fractions containing the rafts were treated with 1% Triton X-100 for 30 min at 37° C. The solubilized fractions were applied to ELISA plates coated with rabbit polyclonal antibodies against integrin β3, blocked with 0.6% casein and then incubated overnight at 4° C. After washing three times with PBS-T, the plate wells were treated with primary goat antibodies (0.2 μg/mL, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 or Hsp40 for 1 h at 37o C. Following three times washing with PBS-T, secondary HRP-conjugated donkey anti-goat IgG (0.133 μg/mL, Santa Cruz Biotechnology Inc. SC-2020) was added, followed by incubation for 1 h at 37o C. After PBS-T washing three times, OPD was used to visualize and quantify the reaction as indicated above.

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: Disminución de la infección por rotavirus con anticuerpos contra Hsp90, Hsp70, Hsp60 o Hsp40. A-E. Las células MCF-7 en medio de cultivo se trataron por separado con una dilución en serie de anticuerpos contra las HSPs indicadas, durante 45 minutos a 4 ºC, se lavaron con DMEM y luego se incubaron con los aislados de rotavirus indicados en los paneles. Después de 12 h.p.i., se detectaron antígenos de rotavirus estructurales mediante un ensayo de inmunocitoquímica. Se utilizaron como control células inoculadas con virus sin pretratamiento con anticuerpos. Los datos se expresaron como porcentaje de células infectadas. Los datos se muestran como porcentajes promedios ± desviación estándar (DS) de tres experimentos independientes realizados por duplicado.

Article Snippet: After centrifugation at 120,000 g for 4 h at 4° C, fractions (500 μL) were collected from the top of the gradient and the fractions containing the rafts were treated with 1% Triton X-100 for 30 min at 37° C. The solubilized fractions were applied to ELISA plates coated with rabbit polyclonal antibodies against integrin β3, blocked with 0.6% casein and then incubated overnight at 4° C. After washing three times with PBS-T, the plate wells were treated with primary goat antibodies (0.2 μg/mL, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 or Hsp40 for 1 h at 37o C. Following three times washing with PBS-T, secondary HRP-conjugated donkey anti-goat IgG (0.133 μg/mL, Santa Cruz Biotechnology Inc. SC-2020) was added, followed by incubation for 1 h at 37o C. After PBS-T washing three times, OPD was used to visualize and quantify the reaction as indicated above.

Techniques: Control, Virus

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: After centrifugation at 120,000 g for 4 h at 4° C, fractions (500 μL) were collected from the top of the gradient and the fractions containing the rafts were treated with 1% Triton X-100 for 30 min at 37° C. The solubilized fractions were applied to ELISA plates coated with rabbit polyclonal antibodies against integrin β3, blocked with 0.6% casein and then incubated overnight at 4° C. After washing three times with PBS-T, the plate wells were treated with primary goat antibodies (0.2 μg/mL, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 or Hsp40 for 1 h at 37o C. Following three times washing with PBS-T, secondary HRP-conjugated donkey anti-goat IgG (0.133 μg/mL, Santa Cruz Biotechnology Inc. SC-2020) was added, followed by incubation for 1 h at 37o C. After PBS-T washing three times, OPD was used to visualize and quantify the reaction as indicated above.

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: After centrifugation at 120,000 g for 4 h at 4° C, fractions (500 μL) were collected from the top of the gradient and the fractions containing the rafts were treated with 1% Triton X-100 for 30 min at 37° C. The solubilized fractions were applied to ELISA plates coated with rabbit polyclonal antibodies against integrin β3, blocked with 0.6% casein and then incubated overnight at 4° C. After washing three times with PBS-T, the plate wells were treated with primary goat antibodies (0.2 μg/mL, Santa Cruz Biotechnology Inc.) against Hsp90, Hsp70, Hsp60 or Hsp40 for 1 h at 37o C. Following three times washing with PBS-T, secondary HRP-conjugated donkey anti-goat IgG (0.133 μg/mL, Santa Cruz Biotechnology Inc. SC-2020) was added, followed by incubation for 1 h at 37o C. After PBS-T washing three times, OPD was used to visualize and quantify the reaction as indicated above.

Techniques: In Vitro, Virus