Phospho smad1 5 9

Western blot analysis was performed as previously described [34 (link)]. MH7A cells were treated with IP lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA) containing protease and phosphatase inhibitor cocktails (GenDEPOT) following manufacturer protocol. Lysates were centrifuged at 16,609× g (Hanil, Incheon, Republic of Korea) and 4 °C for 15 min. Protein concentrations were measured using at Bicinchoninic Acid Protein Assay Kit (Thermo Scientific). Samples were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Darmstadt, Germany). Membranes were incubated overnight at 4 °C with primary antibodies phospho-Smad2/3 (1:1000, Cell Signaling, Danvers, MA, USA), phospho-Smad1/5/9 (1:1000, Cell Signaling), LC3B (1:1000; Cell Signaling), p62 (1:1000; Cell Signaling) and Beclin-1 (1:1000; Cell Signaling). Next, they were incubated with HRP-conjugated anti-rabbit (1:50,000; ENZO Life Science, Farmingdale, NY, USA) secondary antibodies for 1 h. An enhanced chemiluminescence kit (Amersham Pharmacia, Piscataway, NJ, USA) and ABI680 Analyzer (Amersham) were used to detect protein bands; signals were quantified in Image J. The loading control was β-actin antibody (1:5000; Sigma-Aldrich).

Lipofectamine 3000 transfection reagent, TRIzol LS, and TGFβ1 were purchased from Invitrogen. Phospho-Smad1/5/9, Smad1, Smad2, Smad3, Smad4, Smad7, and Smad9 antibodies were from Cell Signaling Technology. E-Cadherin, α-Catenin, N-cadherin, Vimentin, Snail1, and HIF1α antibodies were from Abcam. Sox2, Oct4, CD133, and β-tubulin antibodies were from Millipore. Melatonin was purchased from Sigma. CCL20 was obtained from Peprotec. Smad1 and Smad7 plasmid constructs were purchased from Addgene.

Cells or cartilaginous tissue pellets were lysed in lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100 and 1:100 Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) for 5 min on ice, with a preliminary grinding step (in case of pellets) using Powerful ball mill Retsch MM400 (2 rounds for 2 min, 30 Hz). The lysates were cleared by centrifugation, and proteins were resolved by SDS-PAGE, blotted onto a nitrocellulose membrane and analyzed by WB. Following antibodies were used: phospho-SMAD1/5/9 (Cell Signaling, 13820), total SMAD1/5 (Abcam ab33902 and Abcam ab40771), SOX9 (Millipore AB5535), KLF15 (Santa Cruz sc-393627), phospho-ERK1/2 (Santa Cruz sc-7383), total ERK1/2 (Cell Signaling #9102), β-catenin (BD Transduction Laboratories, 610153), active β-catenin (Merck Millipore 05-665), and α-tubulin (Thermo Fisher Scientific, MS-581-P0).

Protein was isolated from chondrocytes from mouse tibial and femoral growth plate or metacarpal and phalangeal growth plate using RIPA buffer supplemented with proteinase inhibitor cocktail (Sigma-Aldrich) and PhosSTOP (Sigma-Aldrich). Cell lysates were incubated on ice for 30 minutes, followed by a 10-minute centrifugation at 21,000 g to remove cell debris. Western blotting was performed as previously described [42 (link)] using either phospho-SMAD1/5/9 (Cell Signaling, number 13820) or total SMAD1/5/9 (Abcam, ab80255).

Smad1/5/9 antibody (1:1000) was obtained from Abcam (ab80255, Cambridge, MA, USA). All other antibodies, including SCD-1 (#2794, 1:1000), phospho-Smad1/5/9 (#13820, 1:1000) and β-actin (#4970, 1:5000), were obtained from Cell Signaling Technology (Beverly, MA, USA). The gene-specific siRNAs, including control (AM4615), Smad1 (HSS106248), Smad5 (HSS106259), PPARα (s10882), PPARδ (s10885) and PPARγ (s10888), were obtained from Thermo (Waltham, MA, USA). The specific siRNA for SCD-1 (SG00327274) were obtained from Sigma-Aldrich (St Louis, MO, USA). All other chemicals of reagent grade were obtained from Sigma-Aldrich (St Louis, MO, USA).

Human recombinant TGF-β1 and TGF-β2 were purchased from Gibco (Carlsbad, CA) and human recombinant BMP4 was purchased from R&D systems (Inc., Minneapolis, MN, USA). The following antibodies were used for Western blotting and immunofluorescence: BMP4 (Thermo Scientific, Carlsbad, CA), α-Smooth muscle and fibronectin (Sigma-Aldrich, MO, USA), E-cadherin (BD biosciences, San Jose, CA), BMPR1A, BMPR1B, activin receptor type IA, Vimentin, smad1/5/9 and β-actin (Abcam Ltd., Cambridge, USA), ZO-1 (Invitrogen, Carlsbad, CA), phospho-smad1/5/9, phospho-Smad2/3 and Smad2/3 (Cell Signaling Technology, Danvers, MA, USA).