Figure 1 Western Blot Validation of MyD88 in HeLa (A) and Jurket (B) Cells
Loading: 15 μg of lysates per lane.
Antibodies: 2125 (1 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 in Cell Lines
Loading: 15 μg of lysates per lane.
Antibodies: MyD88 2125 (2 μg/mL), MyD88 2127 (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 Independent Antibody Validation (IAV) via Protein Expression Profile in Human Tissues
Loading: 15 μg of lysates per lane.
Antibodies: MyD88 2125 (2 μg/mL), MyD88 2127 (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 Animal Species Reactivity
Loading: Lysates/proteins at 15 μg per lane.
Antibodies: 2125 (2 μg/mL) or 2127 (2 μg/mL). 1 h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
Figure 5 Validation with MyD88 siRNA Knockdown
HeLa cells were transfected with control siRNAs (lane 1) or MyD88 siRNAs (lane 2)
Loading: 10 μg of HeLa whole cell lysates per lane.
Antibodies: 2125 (2 μg /mL), 1 h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
Figure 6 Immunofluorescence Validation of MyD88
Immunofluorescent analysis of 4% paraformaldehyde-fixed HeLa cells labeling MyD88 with 2125 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing nucleus staining on human testis cells.
Figure 7 Immunohistochemistry Validation of MyD88
Immunohistochemical analysis of paraffin-embedded human heart tissue using anti-MyD88 antibody (2125) at 2 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 8 Immunofluorescence Validation of MyD88 in K562 Cells
Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling MyD88 with 2125 at 10 μg/mL, followed by Goat anti-rabbit IgG secondary antibody at 1/500 dilution (green) and DAPI staining (blue).
Figure 9 KO Validation in Macrophages (Miller et al., 2006)
Bone marrow-derived macrophages from wild type (WT) mice and MyD88 knockout mice were assessed for MyD88 protein expression by anti-MyD88 antibodies. MyD88 expression was detected in WT mice, but not in MyD88 knockout mice.
Figure 10 KO Validation in MyD88-deficient MEF cell line (Burns et al., 2003)
MyD88?/?-deficient MEF cell line was reconstituted by retroviral infection with an empty vector, MyD88, or MyD88s expression vectors. The levels of MyD88 (isoform 1) or MyD88s (isoform 3) were confirmed with anti-MyD88 antibodies and MyD88 expression was not detected in the MyD88-deficient cells.
Figure 11 KO Validation in IECs (Vlantis et al., 2014)
Western blot with anti-MyD88 antibodies on intestinal epithelial cells (IECs) showing efficient deletion of MyD88 and concomitant expression of GFP in MyD88IEC-KO mice, but not in MyD88 knockout mice.
Figure 12 KD Validation in Chondrocytes (Ahmad et al., 2009)
Chondrocytes were transfected with either MyD88 siRNA or control siRNA and analyzed for MyD88 expression by immunoblotting with anti-Myd88 antibodies that confirmed inhibition of the target proteins.
Figure 13 Immunoprecipitation Validation in HEK293 cells (Kawai et al., 2004)
HEK293 cells were transiently transfected with DYKDDDDK-IRF7. Ccell lysates were immunoprecipitated with control rabbit anti-mouse immunoglobulin serum (IgG) or anti-MyD88 (Ab1 and Ab2), followed by immunoblotting with anti-DYKDDDDK.
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