Day 2 hMSC sheets (n = 3 per group) were homogenized in CelLytic MT lysis buffer (Sigma-Aldrich) supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Equal amounts (15 μg) of protein lysates, determined by a standard bicinchoninic acid (BCA) protein assay kit (Pierce, Thermo Fisher Scientific), were subjected to SDS–polyacrylamide gel electrophoresis using 10% NuPAGE Bis-Tris gels (Invitrogen, Thermo Fisher Scientific) and transferred to 0.45-μm polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were blocked with 5% bovine serum albumin in standard Tris-buffered saline, 0.1% Tween 20. The phosphorylation of intracellular SMAD3 and SMAD5 was detected using specific primary antibodies [anti–phospho-SMAD3 (ab52903) and anti–phospho-SMAD5 (ab92698), Abcam, Cambridge, MA], followed by horseradish peroxidase (HRP)–conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA). Subsequently, the blots were stripped (Western Blot Stripping Buffer, Pierce, Thermo Fisher Scientific) and reprobed for the detection of the respective total protein [anti-SMAD3 (ab40854) and anti-SMAD5 (ab40771), Abcam] and loading control [anti–β-actin (A1978), Sigma-Aldrich] with respective HRP-conjugated secondary antibodies (Jackson ImmunoResearch). Bound antibodies were visualized with the enhanced chemiluminescence detection system (Pierce, Thermo Fisher Scientific) on autoradiography film (Thermo Fisher Scientific). The intensity of immunoreactive bands was quantified using ImageJ software (NIH, Bethesda, MD).

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