Phospho smad1 5 9 antibody

Whole cell lysates were prepared from E10.5 embryo head or E9.5 embryo using NP40 lysis buffer (50 mM Tris-HCl, pH7.4, 200 mM NaCl, 2 mM MgCl₂, 1% Nonidet P-40 (NP-40), 1 mM PMSF, 1 μg/ml leupeptin, 2 μg/ml aprotinin, and 1 μg/ml pepstatin). Proteins were separated on Novex 4–20 % Tris-Glycine Gel (Invitrogen) and transferred to PVDF membrane. The membranes were incubated with 5% milk for 1 h and incubated with primary antibodies overnight at 4°C. Primary antibodies were used as follows: phospho-Smad1/5/9 antibody (1:1000, #13820, Cell Signaling), phospho-mTOR (Ser2448) (1:1000, #2971, Cell Signaling), mTOR (1:1000, #2972, Cell Signaling), phospho-S6K1 (Thr389) (1:500, #9205, Cell Signaling), S6K1 (1:1000, #9202, Cell Signaling), phospho-S6 (Ser235/236) (1:2000, #2211, Cell Signaling), phospho-S6 (Ser240/244) (1:2000, #5364, Cell Signaling), S6 (1:2000, #2217, Cell Signaling), TSC1 (1:1000, #6935, Cell Signaling), and β-actin (1:1000, #4970, Cell Signaling). Blots were incubated with peroxidase-coupled secondary antibodies (Promega) for 1 h, and protein levels were detected with SuperSignal West Pico or Femto Chemiluminescent Substrate (Thermo Scientific). Images were quantified by ImageJ software (NIH).

BMP4 transcriptional effects were previously reported to be mediated by phospho-Smad3, which is increased and undergoes nuclear translocation following BMP4 exposure [21 (link)-25 (link)]. NCM460 cells tested included: untreated control, control-silenced by siRNA, ARSB-silenced by siRNA, treated with exogenous human recombinant BMP4 (20 ng/ml × 24 h; R&D, Minneapolis, MN), or exposed to the common food additive carrageenan (1 μg/ml × 24 h) which reduces ARSB activity. The nuclear chromatin from the various cell preparations was prepared and processed, as previously described [2 (link),16 ]. Primers that encompassed the phospho-Smad3 binding site in the CHST11 promoter were selected, and the targeted promoter oligonucleotide (5′-GTCT-3′) was amplified by QRT-PCR. Binding of phospho-Smad3 antibody (Cell Signaling Technology, Danvers, MA) and of phospho-Smad 1,5,9 antibody (Cell Signaling) to the CHST11 promoter oligonucleotide was compared among the different nuclear chromatin preparations, noting that Smad8 is also named as Smad9. Agarose gel electrophoresis was performed to illustrate the variation in phospho-Smad3 binding to the different nuclear preparations and to confirm the lack of binding of the phospho-Smad1,5,9 antibody to the CHST11 promoter oligonucleotide.