- PA5-78853 - Invitrogen Antibodies BAG2 antibody

Chen LJ, Hu B, Han ZQ, Zhu JH, Fan X, Chen XX, Li ZP, Zhou H
Frontiers in cell and developmental biology 2020;8:554190

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry analysis of BAG2 using anti-BAG2 antibody (Product # PA5-78853). BAG2 was detected in an immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum and then incubated with 5 μg/mL rabbit anti-BAG2 antibody (Product # PA5-78853) overnight at 4°C. DyLight®594 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry analysis of BAG2 using anti-BAG2 antibody (Product # PA5-78853). BAG2 was detected in an immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum and then incubated with 5 μg/mL rabbit anti-BAG2 antibody (Product # PA5-78853) overnight at 4°C. DyLight®594 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow Cytometry of BAG2 in THP-1 cells (blue line), isotype control rabbit IgG (green line) and unlabeled (red line). Samples were blocked with 10% goat serum, incubated with BAG2 Polyclonal Antibody (Product # PA5-78853) at a dilution of 1 μg (per 1x10^6 cells), followed by DyLight®488 conjugated goat anti-rabbit IgG (for 30 minutes at 20°C) using 5-10 μg (per 1x10^6 cells) dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow Cytometry of BAG2 in THP-1 cells (blue line), isotype control rabbit IgG (green line) and unlabeled (red line). Samples were blocked with 10% goat serum, incubated with BAG2 Polyclonal Antibody (Product # PA5-78853) at a dilution of 1 μg (per 1x10^6 cells), followed by DyLight®488 conjugated goat anti-rabbit IgG (for 30 minutes at 20°C) using 5-10 μg (per 1x10^6 cells) dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of BAG2 in THP-1 cells using BAG2 Polyclonal Antibody (Product # PA5-78853), shown in overlay histogram (blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum, and incubated with the primary antibody (1 μg/1x10^6 cells) for 30 min at 20°C. DyLight 488 conjugated goat anti-rabbit IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10^6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Supportive validation

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 4 BAG2 inhibits CHIP-mediated degradation of ERbeta in HESCs in vitro . (A) The mRNA expression of BAG2 in human and mouse endometriosis/normal tissues and HESCs determined by RT-qPCR. (B) The protein expression of BAG2 in human and mouse endometriosis/normal tissues and HESCs determined by Western blot analysis. (C) The statistical analysis of immunohistochemical staining of BAG2 in human and mouse endometriosis/normal tissues. (D) The interaction between CHIP and BAG2 by Co-IP assay. (E) Ubiquitin of ERbeta in the presence or absence of BAG2 by ubiquitin test. (F) The protein expression of ERbeta in the presence or absence of BAG2 by Western blot analysis. (G) The statistical analysis of panel (F) . Comparisons between two groups were analyzed using independent sample t -test. * p < 0.05 vs. human or mouse normal tissues, HESCs-NC cells or the absence of BAG2. n = 78 for human normal tissues; n = 91 for human endometriosis tissues; n = 14 for mouse endometriosis/normal tissues. Cell experiments were conducted in triplicate.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 6 BAG2-mediated inhibition of ERalpha and MDM2-mediated elevation of ERbeta contributes to the development of endometriosis. HESCs-NC cells were treated with overexpressed MDM2 and BAG2 with oe-NC as control, while HESCs-En cells were treated with sh-BAG2 and sh-MDM2 with sh-NC as control. (A) The mRNA expression of BAG2 and MDM2 determined by RT-qPCR. (B) The protein expression of BAG2 and MDM2 determined by western blot analysis. (C) The protein expression of ERalpha and ERbeta determined by western blot analysis. (D) The protein expression of ERbeta/ERalpha determined by western blot analysis. Comparisons between two groups were analyzed using independent sample t -test; * p < 0.05 vs. oe-NC treatment; # p < 0.05 vs. sh-NC treatment. Cell experiments were conducted in triplicate.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 7 The interference of BAG2 and MDM2 showed therapeutic effects on mouse endometriosis. Con-M mice were treated with overexpressed MDM2 and BAG2 using oe-NC as control, while En-M mice were treated using sh-BAG2 and sh-MDM2 with sh-NC as control. (A) The mRNA expression of BAG2 and MDM2 determined by RT-qPCR. (B) The protein expression of BAG2 and MDM2 determined by Western blot analysis. (C) The protein expression of ERalpha determined by Western blot analysis. (D) The protein expression of ERbeta determined by Western blot analysis. (E) The protein expression of ERbeta/ERalpha determined by Western blot analysis. (F) Fluorescence staining of Ki67 in mouse tissues. G, The statistical analysis of (F) . (H) Immunohistochemical staining of cleaved caspase 8 in mouse tissues. (I) The statistical analysis of panel (H) . Comparisons between two groups were analyzed using independent sample t -test. n = 8. * p < 0.05 vs. oe-NC treatment; # p < 0.05 vs. sh-NC treatment.

PA5-78853