Anti SMAD3, phospho Ser423, phospho Ser425

Rockland's affinity purified anti-Smad3 pS423 pS425 antibody was used at 2.5 ug/ml to detect signal in a variety of tissues including multi-human, multi-brain and multi-cancer slides. This image shows strong nuclear staining in the majority of epidermal keratinocytes at 40X. Tissue was formalin-fixed and paraffin embedded. The image shows localization of the antibody as the precipitated red signal, with a hematoxylin purple nuclear counterstain. Personal Communi-cation, Tina Roush, LifeSpanBiosciences, Seattle, WA.

Western Blot of Rabbit anti-SMAD3 pS423 pS425 antibody.
Marker: Opal Pre-stained ladder (p/n MB-210-0500).
Lane 1: HEK293 lysate (p/n W09-000-365).
Lane 2: HeLa Lysate (p/n W09-000-364).
Lane 3: MCF-7 Lysate (p/n W09-000-360).
Lane 4: Jurkat Lysate (p/n W09-000-370).
Lane 5: A431 Lysate (p/n W09-000-361).
Lane 6: A549 Lysate (p/n W09-001-372).
Lane 7: LNCap Lysate (p/n W09-001-GJ9).
Lane 8: MOLT-4 Lysate (p/n W09-001-GK2).
Lane 9: Ramos Lysate (p/n W09-000-GK4).
Lane 10: Raji Lysate (p/n W09-001-368).
Lane 11: A-172 Lysate (p/n W09-001-GL5).
Lane 12: NIH/3T3 Lysate (p/n W10-000-358).
Load: 10 μg per lane.
Primary antibody: SMAD3 pS423 pS425antibody at 1ug/mL overnight at 4C.
Secondary antibody: Peroxidase rabbit secondary antibody (p/n 611-103-122) at 1:30,000 for 60 min at RT.
Blocking Buffer: 1% Casein-TTBS (p/n MB-082) for 30 min at RT.
Predicted/Observed size: 35 kDa for SMAD3 pS423 pS425.

Western blot using Rockland's affinity purified anti-Smad3 pS423 pS425 antibody shows detection of endogenous Smad3 in stimulated cell lysates. Lysates were prepared from control cells (- lanes), or cells stimulated with 2 ng/ml TGF (+lanes) for 1 hour. This reagent recognizes phosphorylated Smad3 and has negligible reactivity against non-phosphorylated Smad3 protein. Personal Communication. Ying Zhang, NIH, CCR, Bethesda, MD.