Figure 1 Western Blot Validation in Cell Lines
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), beta-actin (1 μg /mL), and GAPDH (0.02 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 in HeLa Cells
Immunofluorescent analysis of 4% paraformaldehyde-fixed HeLa cells labeling DR4 with 1167 at 5 μg /mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue). Image showing membrane staining on HeLa cells.
Figure 4 Immunocytochemistry Validation of DR4 in HeLa Cells
Immunocytochemical analysis of 4% paraformaldehyde-fixed HeLa cells labeling DR4 with 1167 at 2 μg /ml, followed by goat anti-rabbit IgG secondary antibody at 1/250 dilution (red). Image showing membrane staining on HeLa cells.
Figure 5 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 was disrupted. Dipyridamole up-regulated the expression of DR4.
Figure 6 Immunofluorescence Validation of DR4 in Rat Brain (Cantarella et al., 2014)
DR4 protein expression detected by anti-DR4 antibodies 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 7 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 or anti-DR5, AEC, hematoxylin). Both DR4 and DR5 expression were upregulated in MeWo cells after treatment.
Figure 8 Immunohistochemistry Validation of DR4 in Human Colon Tumors (Devetzi et al., 2016)
DR4 expression in human colon tumors detected by anti-DR4 antibodies. Strong immunoreactivity is shown for DR4 in T167. Moderate immunoreactivity is shown for DR4 in T187.
Figure 9 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 μM luteolin for 24 h. Western blot analysis was carried out with anti-DR4 antibodies. DR4 expression was markedly reduced after DR4 knockdown.
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