Anti-PUMA BBC3 Antibody

Western Blot Validation of PUMA in K562 Cells
Loading: 15 μg of lysates per lane. Antibodies: A04899-1 (2 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution

Independent Antibody Validation (IAV) via Protein Expression Profile in Human Cells
Loading: 20 μg of lysates per lane. Antibodies: 3041 (3 μg/mL), A04899-1 (2 μg/mL), beta-actin (1 μg/mL) and GAPDH (0.02 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.

Immunofluorescence Validation of PUMA in K562 Cells
Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling PUMA with A04899-1 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue).

Immunohistochemistry Validation of PUMA in Human Breast Carcinoma
Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using anti-PUMA antibody (A04899-1) at 10 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

Immunohistochemistry Validation of PUMA in Human Breast Tissue
Immunohistochemical analysis of paraffin-embedded human breast tissue using anti-PUMA antibody (A04899-1) at 2.5 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

Immunofluorescence Validation of PUMA in K562
Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling PUMA with A04899-1 at 10 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing cytosol staining on K562 cells

Induced Expression of PUMA in MCF7 cells (Wade et al., 2008)
Western analysis of MCF7 treated with the indicated dose of Nutlin-3a or ABT-737 for 24h. Note that Puma is induced following Nutlin-3a treatment in these cells and PUMA expression was detected by anti-PUMA antibodies (A04899-1)

KO Validation of PUMA in Mouse Thymocytes (Michalak et al., 2008)
Western blot analysis of thymocytes from wt, noxa knockout, puma knockout and noxa/puma double knockout mice cultured for 7 h in the presence or absence of 2.5 Gy g-irradiation. PUMA expression was not detected in puma KO and double KO mice with anti-PUMA antibodies (A04899-1).

KO Validation of PUMA in Mouse Cerebellar Neurons (Ambacher et al., 2012)
Puma expression is induced by potassium withdrawal in cerebellar granule neurons. After 7 days in culture CGNs were either maintained in media containing 25 mM potassium (K25) or switched to low potassium medium containing 5 mM potassium (K5). PUMA protein levels were analyzed by western blot with anti-PUMA antibodies (A04899-1). PUMA expression was not detected in PUMA KO mice and was increased after treatment in WT.

Immunofluorescence Validation of PUMA in Rat Retina (Wakabayashi et al., 2012)
PUMA expression in the rat retina detected by anti-PUMA antibodies (A04899-1). The specimens were counterstained with Hoechst 33258 to visualize nuclei (+DNA). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; P, postnatal day.