Figure 1 Western Blot Validation of PUMA in K562 Cells
Loading: 15 μg of lysates per lane.
Antibodies: 3043 (2 μg/mL), 1 h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution
Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile
Loading: 20 μg of lysates per lane.
Antibodies: 3041 (3 μg/mL), 3043 (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.
Figure 3 Immunofluorescence Validation of PUMA
Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling PUMA with 3043 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue).
Figure 4 Immunohistochemistry Validation of PUMA
Immunohistochemistry analysis of 4% paraformaldehyde-fixed human breast carcinoma cells labeling PUMA with 3043 at 10 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution. Image showing cytosol staining in the cancer cells.
Figure 5 Immunohistochemistry Validation of PUMA
Immunohistochemical analysis of 4% paraformaldehyde-fixed human breast tissue labeling PUMA with 3043 at 2.5 μg/ml, followed by goat anti-rabbit IgG secondary antibody at 1/250 dilution. Image showing both cytosol staining on K562 cells.
Figure 6 Immunofluorescence Validation of PUMA
Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling PUMA with 3043 at 10 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing cytosol staining on K562 cells
Figure 7 KO Validation of PUMA (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 (3043).
Figure 8 KO Validation of PUMA (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 (3043). PUMA expression was not detected in PUMA KO mice and was increased after treatment in WT.
Figure 9 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 (3043)
Figure 10 Immunofluorescence images of PUMA in the P14 rat retina (Wakabayashi et al., 2012)
PUMA expression in the rat retina detected by anti-PUMA antibodies (3043). 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.
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