Phospho-IRF3 (S396) Recombinant Rabbit Monoclonal Antibody [PSH06-75]

☑ Cell treatment (CT)

Western blot analysis of Phospho-IRF3 (S396) on different lysates with Rabbit anti-Phospho-IRF3 (S396) antibody (HA722772) at 1/1,000 dilution.

Lane 1: HT-29 untransfected cell lysate
Lane 2: HT-29 transfected with 2.5μg/mL Poly(I:C) for 6 hours cell lysate
Lane 3: J774A.1 untransfected cell lysate
Lane 4: J774A.1 transfected with 2.5μg/mL Poly(I:C) for 6 hours cell lysate
Lane 5: HT-29 transfected with 2.5μg/mL Poly(I:C) for 6 hours cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 47 kDa
Observed band size: 47-55 kDa

Exposure time: 12 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722772) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Cell treatment (CT)

Immunohistochemical analysis of paraffin-embedded human testis tissue untreated / treated with λpp with Rabbit anti-Phospho-IRF3 (S396) antibody (HA722772) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA722772) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

☑ Cell treatment (CT)

Immunocytochemistry analysis of untreated HT-29 cells (top) / HT-29 cells transfected with 2.5μg/mL Poly(I:C) for 6 hours (bottom) labeling Phospho-IRF3 (S396) with Rabbit anti-Phospho-IRF3 (S396) antibody (HA722772) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-IRF3 (S396) antibody (HA722772) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

☑ Cell treatment (CT)

Flow cytometric analysis of untreated HT-29 cells (top) / HT-29 cells transfected with 2.5μg/mL Poly(I:C) for 6 hours (bottom) labeling Phospho-IRF3 (S396).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722772, 1/1,000) (right) compared with Rabbit IgG Isotype Control (left). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃.