Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y](AB52903)

TGF-β1 signaling is impaired in NDRG1-silenced MEFs. PML+/+ mouse embryonic fibroblasts (MEFs) were transfected with either CTL-siRNAs (A & B) or NDRG1-siRNAs (C & D) and induced with100 ng/ml TGF-β1. Immunofluorescent staining revealed intense nuclear staining for phosphorylated SMAD3 (SMAD3-P, ab52903) in CTL-siRNA treated MEFs (B) while only weak nuclear staining for MEFs treated with NDRG1-siRNA (D).

Image from Tang MK et al. PLoS One. 2013;8(3):e59477. Fig 12.; doi: 10.1371/journal.pone.0059477.

Blocking and diluting buffer: 5% NFDM/TBST.

All lanes:

Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) at 1/2000 dilution

Lane 1:

F9 (Mouse embryonic testicular cancer epithelial cell) whole cell lysates at 15 µg

Lane 2:

F9 (Mouse embryonic testicular cancer epithelial cell) whole cell lysates. Then the membrane was incubated with phosphatase. at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 48 kDa

Observed band size: 50 kDa

Exposure time: 1min

Representative IHC photomicrographs from an Environmental enteropathy (EE) duodenal biopsy showing p-SMAD3 staining (ab52903) in only the epithelium (arrows).

Image from Syed S et al. PLoS Negl Trop Dis. 2018;12(2):e0006224. Fig 4.; doi: 10.1371/journal.pntd.0006224.

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes:

Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) at 1/1000 dilution

Lane 1:

A549 whole cell lysate at 10 µg

Lane 2:

A549 treated with 5ng/ml TGF-β1 for 24 hours whole cell lysate at 10 µg

Lane 3:

A549 treated with 5ng/ml TGF-β1 for 24 hours whole cell lysate, the membrane was incubated with alkaline phosphatase at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 48 kDa

Observed band size: 55 kDa

All lanes:

Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) at 1/2000 dilution

Lane 1:

(A) HL-60 cell lysates at 10µg untreated

Lane 2:

(B) HL-60 cell lysates at 10µg treated with TGF.

Predicted band size: 48 kDa

Observed band size: 45 kDa, 55 kDa