Figure 1 Western Blot Validation in Human, Mouse and Rat Cell Lines
Loading: 15 ug of lysates per lane.
Antibodies: JMJD3, (1 ug/mL), 1h 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 ug of lysates per lane.
Antibodies: JMJD3 (1 ug/mL), JMJD3 14-618, (2 ug/mL) and beta-actin 3779 (1ug/mL), 1h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
Figure 3 Western Blot Validation in HeLa Cell Lysate
Loading: 15 ug of lysates per lane.
Antibodies: JMJD3, (0.25 ug/mL), 1h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
Figure 4 Western Blot Validation in K562 Cell Lysate
Loading: 15 ug of lysates per lane.
Antibodies: : JMJD3, (0.5 ug/mL) , 1h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
Lane 1: in the absence and Lane 2: in the presence of blocking peptide
Figure 5 Western Blot Validation in Mouse A-20 Cell Lysate
Loading: 15 ug of lysates per lane.
Antibodies: JMJD3, (0.5 ug/mL), 1h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
Lane 1: in the absence and Lane 2: in the presence of blocking peptide
Figure 6 Western Blot Validation in HepG2 Cell Lysate
Loading: 15 ug of lysates per lane.
Antibodies: JMJD3, 1h incubation at RT in 5% NFDM/TBST.
Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
Lane 1: 0.25 ug/mL Lane 2: 0.5 ug/mL Lane 3: 0.25 ug/mL with blocking peptide
Figure 7 Immunocytochemistry Validation of JMJD3 in K562 Cells Immunocytochemical analysis of K562 cells using anti- JMJD3 antibody (MBS150629) at 2.5 ug/ml. Cells 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 JMJD3 in K562 Cells Immunofluorescent analysis of 4% paraformaldehyde fixed K562 cells labeling JMJD3 with MBS150629 at 20 ug/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
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