Figure 1 Western Blot Validation in Human Cell Lines
Loading: 15 μg of lysates per lane.
Antibodies: DR4 1139 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
Lane A: HepG2 cell lysate
Lane B: Jurkat cell lysate
Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Cell Lines
Loading: 15 μg of lysates per lane.
Antibodies: DR4 1139 (1 μg/mL), DR4 1167 ( 4 μg/mL), and beta-actin (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
Figure 3 Immunofluorescence Validation of DR4
Immunofluorescent analysis of 4% paraformaldehyde-fixed human spleen tissue labeling DR4 with 1139 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue). Image showing membrane staining on human spleen cells.
Figure 4 Immunocytochemistry Validation of DR4
Immunocytochemical analysis of 4% paraformaldehyde-fixed Jurkat cells labeling DR4 with 1139 at 10 μg/ml, followed by goat anti-rabbit IgG secondary antibody at 1/250 dilution (red). Image showing membrane staining on Jurkat cells.
Figure 5 Immunohistochemistry Validation of DR4
Immunohistochemical analysis of paraffin-embedded human spleen tissue using anti-DR4 antibody (1139) 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.
Figure 6 KD Validation in SW480 cells (Goda et al., 2008)
The expression of DR4 was knocked down via DR4 siRNA, 24 h later
cells were treated with dipyridamole for 24 h. DR4 protein expression detected by anti-DR4 antibodies (1139) was disrupted. Dipyridamole up-regulated the expression of DR4.
Figure 7 Immunofluorescence Validation of DR4 in rat brain (Cantarella et al., 2014)
DR4 protein expression detected by anti-DR4 antibodies (1139) was increased after transient brain ischemia (tMCAO) and decreased after pre-conditioning stimulus. Confocal microscopic images displaying NeuN (a,d, g) (green), DR4 (b, e, h) (red), and Merge (c, f, i) (yellow) in the brain peri-ischemic region of rats after 5 h.
Figure 8 Immunocytochemistry Validation of DR4 in human melanoma cells (Ekmekcioglu et al., 2008)
MeWo melanoma cells were exposed to affinity-purified MDA7/IL-24. After 48 h of treatment, cells were collected and cytospins prepared for cytochemical assessment of their TRAIL receptor (R1 and R2) expression (anti-DR4 (1139) or anti-DR5, AEC, hematoxylin). Both DR4 and DR5 expression were upregulated in MeWo cells after treatment.
Figure 9 KD Validation in Huh7 cells (Malhi et al., 2007)
Western blot analysis with anti-DR4 antibodies (1139) was performed for DR5 and DR4 expression using whole cell lysates from Huh 7 cells transfected with respective siRNAs. In cells treated with siDR4, a decrease in DR4 level was observed, DR5 levels were unchanged. Scrambled siRNA was used as control.
Figure 10 KD Validation in HeLa cells (Horinaka et al., 2005)
HeLa cells were transfected with DR4
siRNA or LacZ control siRNA. At 24 h after transfection, the cells were treated with or without 20 ?M luteolin for 24 h. Western blot analysis was carried out with anti-DR4 antibodies (1139). DR4 expression was markedly reduced after DR4 knockdown.
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