Phospho smad1 5 8

Cells were lysed in NETN lysis buffer (100 nM Tris-Cl [pH 7.8], 1 mM EDTA, 100 mM NaCl, and 0.1% Triton X-100) with protease and phosphatase inhibitors (Roche). Protein concentration was determined by Lowry protein assay. Protein samples were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were incubated with antibodies directed against phospho-SMAD1/5/8 (Cell Signaling), total SMAD1/5/8 (Cell Signaling), p63 (4A4 clone; Lab Vision), p53 (Clone DO-7; Thermo Scientific), and β-Actin (Cell Signaling) primary antibodies. Horseradish peroxidase (HRP) conjugated secondary antibodies were used. Blots were visualized by enhanced chemi-luminescence (Millipore).

Tissue collection, 4% paraformaldehyde fixation, processing, paraffin embedding, whole mount staining for β-galactosidase, and H&E staining were performed using established protocols (Lindsley et al., 2007 (link); Snider et al., 2008 (link), 2009 (link)). Collected tissues were sectioned at 6μm thickness. Immunohistochemistry was performed using ABC kit (Vectorstain) with DAB and hydrogen peroxide as chromogens, as described (Olaopa et al., 2011 (link)). The following primary antibodies were used: phospho-SMAD1/5/8 (1:40000, Cell Signaling), α-Smooth muscle actin (1:5000, Sigma), MF20 (1:100, Developmental Studies Hybridoma Bank) and PECAM-1 (1:200, BD Biosciences Pharmingen). Both MF20 and phospho-SMAD1/5/8 antibodies required 10min antigen retrieval (DAKO). Antibody diluent (DAKO), without primary antibody, was used for negative controls. For each assay, whole embryos and/or serial sections were examined for at least three individual embryos of each genotype at each stage of development. Wildtype littermates and Nkx2.5^Cre only embryos were always used as age-matched control samples.

Cells and mouse tissues were lysed in lysis buffer [tris-buffered saline (TBS) containing 1% NP-40, 1 µM NaF, and 1 mM EDTA). The frontal cortexes of FXS patients and controls were lysed in RIPA (radioimmunoprecipitation assay) buffer. Lysed samples were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane (Millipore), immunoblotted with antibodies, and visualized using a LI-COR Imaging System. The antibodies used were directed against FLAG epitope tag (M2, Sigma), FMRP (ab17722, Abcam), BMPR2 KD (19087-1-AP, Proteintech Group), total SMAD1/5/8 (BMR 00479, Bio Matrix Research), phospho-SMAD1/5/8 (#9511, Cell Signaling), cofilin (#5175, Cell Signaling), phospho-cofilin (SC-12912-R, Santa Cruz), β-actin (A5441, Sigma), and GAPDH (MAB374, Millipore).

Equal amounts of protein were heated to 70°C for 10 minutes with NuPAGE LDS Sample Buffer (4×) (Life Technologies), ran on NuPAGE Novex 4–12% Bis-Tris gels (Life Technologies), and transferred to polyvinylidene difluoride membranes (Life Technologies). Membranes were blocked for one hour at room temperature with 5% nonfat milk in Tris Buffered Saline with Tween (TBST; 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.1% Tween 20). Following this, membranes were incubated with primary antibody overnight in blocking solution at 4°C with slight agitation. Primary antibodies for BMP7 (Abcam), phospho-Smad1/5/8 (Cell Signaling), Smad1 (Cell Signaling) and β-tubulin (Cell Signaling) were diluted according to manufacturer’s recommendations. The membranes were washed with TBST and incubated with secondary antibody for one hour at room temperature. The secondary antibody (polyclonal goat anti-rabbit immunoglobulins/horseradish peroxidase, ThermoFisher) was diluted according to manufacturer’s recommendations in blocking solution. Membranes were washed with TBST and developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific). Experiments were performed in triplicate, and .tiff images were analyzed using ImageJ (https://imagej.nih.gov/ij/). Bands were quantified and intensities were normalized with β-tubulin.

After the indicated duration of treatment, cells were lysed in phosphate buffered saline (PBS) containing 50mM Tris pH 8.0, 1% igepal, 0.1% SDS, 0.5% sodium deoxycholate and Roche complete protease inhibitor cocktail (Roche, Basel, Switzerland). Lysates were sonicated and frozen at −80°C until used. Cell lysates (20–40μg total protein) were separated on reducing SDS-PAGE gels and proteins were transferred to polyvinylidene fluoride membranes by semi-dry blotting. Membranes were then blocked and probed with rabbit polyclonal antibodies towards JNK (catalogue # 9252), phospho-JNK (catalogue # 9251), Smad1 (catalogue # 9743), phospho-Smad1/5/8 (catalogue # 9511, all Cell Signaling Technology, Danvers, MA) or PCNA (catalogue # ab18197, Abcam, Cambridge, UK), rabbit monoclonal antibodies towards Caspase-3 (clone 8G10), cleaved Caspase-3 (clone 5A1E), phosphorylated Smad1/5 (clone 41D10, all Cell Signaling Technology, Danvers, MA), Smad5 (clone EP619Y, Epitomics, Burlingame, CA), Id1 (clone 195-14) or Id3 (clone 17-3, both CalBioreagents, San Mateo, CA) or a mouse monoclonal antibody towards BMPR-II (clone 18/BMPR-II, BD Transduction Laboratories, Franklin Lakes, NJ). As a loading control, all blots were re-probed with a monoclonal antibody towards either α-tubulin (clone DM1A) or β-actin (clone AC-15, Sigma). Densitometry was performed using ImageJ software.