Rabbit anti phospho smad2 ser465 467 smad3 ser423 425

To assess the level of Smad2/3 phosphorylation, fibroblasts were lysed using RIPA buffer (1% NP40, 10 mM Tris-HCl pH 7.4, 5 mM EDTA, 150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 2 mM sodium orthovanadate, 10 mM sodium fluoride) supplemented with protease and phosphatase inhibitors (Sigma). Samples were denatured by boiling for 5 min at 95° degrees in Laemmli buffer and separated by SDS-Page using a 10% bis-tris acrylamide gel. Proteins were blotted on a nitrocellulose membrane using the semi-dry Power Blotter system (Invitrogen). Membranes were blocked in 5% BSA (for pSMAD2/3) or 5% skimmed milk (for total SMAD2/3) in TBS 0.1% Tween20 (TBST) for 30 min at room temperature, then incubated overnight at 4 °C with rabbit anti-Smad2/3 (Cell Signaling 8685) or rabbit anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (Cell Signaling 8828), diluted 1:1000 in their blocking solution. After five washes with TBST, membranes were incubated with goat anti-rabbit HRP-conjugated antibody (DAKO, p0448), diluted in their blocking solution 1:2000 for 1 h at room temperature. After five washes with TBST, membranes were incubated with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermoscientific) and chemiluminescence was detected using Chemidoc Touch imaging system (Promega).

Cells were homogenized in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton-X 100, and 1 mM ethylenediaminetetraacetic acid (EDTA) containing protease inhibitor (Roche). The lysates were clarified by centrifugation at 8000 g at 4°C for 20 min, and the supernatants were collected and normalized for protein concentration. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). After blocking with PBS containing 5% skim milk and 0.05% Tween 20, the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with a fluorescently-labeled secondary antibody for 1 hr at room temperature. The following antibodies were used: rabbit anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (8828, Cell Signaling Technology) and rabbit anti-Smad2/3 (D7G7) (8685, Cell Signaling Technology). Horseradish peroxidase–conjugated anti-rabbit IgG antibody was used as secondary antibody (Cell Signaling Technology).
Immunoreactive bands were detected using a fluorescence-conjugated secondary antibody and an enhanced chemiluminescence (ECL) system (WBKLS0100, Millipore), and visualized on a LAS-4000 imaging system (Fujifilm). The protein bands were quantified using the ImageJ software.

For TβRIII RNAi identification, proteins were extracted from MSCs 72 h after transfection. For detection of Smad2/3 phosphorylation, proteins were extracted from pellets after 24 h of chondrogenic induction. The protein concentration was quantified by a BCA Protein Assay Kit (CWBio, Beijing, China). 100 μg proteins was subjected to 6% SDS-PAGE and electrotransferred onto PVDF membrane (Millipore, Boston, USA) at 250 mV for 100 min. After blocking with 5% skim milk and Tris-buffered saline containing 0.1% Tween-20, the PVDF membranes were incubated with antibodies against human TβRIII antibody (Santa Cruz, Dallas, USA), rabbit anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (Cell Signaling, Danvers, USA), rabbit anti-Smad2/3 (Cell Signaling, Danvers, USA), and anti-GAPDH monoclonal antibody (EarthOx, SFO, USA) followed by incubation with HRP-conjugated corresponding secondary antibodies. The signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce, NY, USA). Protein levels in phosphorylated-Smad2/3 (P-Smad2/3) were normalized to those of total Smad2/3 quantities or GAPDH.