Figure 1 Western Blot Validation of PUMA in K562 and 3T3/NIH Cells
Loading: 15 μg of lysates per lane.
Antibodies: 3041 (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 Validation with PUMA siRNA Knockdown
293 cells were transfected with control siRNAs (lane 1) or PUMA siRNAs (lane 2)
Loading: 15 μg of 293 whole cell lysates per lane.
Antibodies: 3041 (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 4 Sensitivity Test for PUMA
Loading: Lysates/proteins at 15 μg per lane.
Antibodies: 3041 (lane 1-3: 1, 2 and 4 μg/mL). 1 h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
Figure 5 Immunofluorescence Validation of PUMA
Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling PUMA with 3041 at 2 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing cytosol staining on K562 cells.
Figure 6 Immunocytochemistry Validation of PUMA
Immunocytochemical analysis of 4% paraformaldehyde-fixed K562 cells labeling PUMA with 3041 at 1 μg/ml, followed by goat anti-rabbit IgG secondary antibody at 1/250 dilution. Image showing both cytosol staining on K562 cells.
Figure 7 KD Validation of PUMA (Elgendy et al., 2011)
HOSE-RasV12 cells were transfected with control shRNA plasmid or shRNA plasmids (KD) targeted against Noxa or Puma, as indicated. PUMA expression was not observed in PUMA KD cells detected by anti-PUMA antibodies (3041).
Figure 8 Induction Validation of PUMA (Sabirzhanov et al., 2014)
PUMA protein levels were increased in etoposide-treated primary cortical neurons detected by anti-PUMA antibodies (3041).
Figure 9 KD Validation of PUMA (Han et al., 2010)
Immunoblot analyses of Tet-induced p53 cells treated with NOXA, Puma, Bim or non-targeting siRNAs that were utilized in this experiment. PUMA protein levels were markedly reduced in PUMA KD cells detected by anti-PUMA antibodies (3041).
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