Phospho smad2 s465 467

Western blot analysis was performed largely as described [53 (link)]. Animal caps (10–15 per condition) were lysed in 5ul/animal cap of lysis buffer (150 mM NaCl, 20 mM Tris pH 7.5, 1% Nonidet P-40, 1 mM EDTA, and 1 protease and phosphatase inhibitor tablet/10 ml of buffer) (Thermofisher) [53 (link)]. After incubation on ice for 30 min, animal cap lysates were centrifuged at 4 °C for 5 min at 14,000 g. Clear supernatant was retained. 5–15 ul of supernatant was run for each sample. Antibodies against phospho-Smad1/5 (S463/465) (Cell Signaling Technology), Smad1 (Cell Signaling Technology), Phospho-Smad2 (S465/467) (Cell signaling technology), Smad2/3 (Cell signaling technology) and GAPDH (Sigma) were all used at 1:1000 dilution. Secondary antibodies (donkey anti-rabbit IgG, or donkey anti-mouse IgG) coupled to horseradish peroxidase (Jackson ImmunoResearch) were used at 1:10,000 dilution. Bands were subjected to densitometric analysis and graphed.

Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN). Secondary antibodies used were: goat anti-mouse IgG horse radish peroxidase (HRP) conjugate and goat anti-rabbit IgG HRP conjugate, both from Calbiochem (EMD Biosciences, La Jolla, CA). Recombinant human VEGF₁₆₅ was purchased from R&D Systems (Minneapolis, MN). Recombinant Human TGFβ2 (100-35B) was from Peprotech (Quebec, QC). Avastin® (DIN 02270994) was from Roche (Mississauga, ON) and was used at a concentration of 1 μg/ml.

Antibodies included: β1 integrin (#610167), β4 integrin (#553745), α6 integrin (#555734) (all from BD Biosciences); Smad2 (#3103), phospho-Smad2 (S465/467, #3101), NF-κB p65 (#4764), phospho-NF-κB p65 (S536, #3033), E-cadherin (#4065), β-actin (#4967), Caspase-3 (#9662), Cleaved Caspase-3 (#9661), Cleaved Caspase-9 (#9509), cleaved PARP (#9544) (all from Cell Signaling), α-tubulin (#T5168, Sigma-Aldrich), ZO-1 (#sc-33725, Santa Cruz), and Alexa-Fluor®-conjugated secondary antibodies (Molecular Probes®, Life Technologies). Growth factors/hormones included: rhTGFβ1 (Invitrogen) and insulin (Sigma-Aldrich). The TβRI inhibitor SB-431542 was from InvivoGen.