Phospho smad1 5 8 antibody

Total RNA was extracted from whole tracheas using the Direct-zol RNA MiniPrep Kit (Zymo Research). cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad), and quantitative RT-PCR was performed with iQ SYBR Green Supermix (Bio-Rad) using a StepOne Plus system (Applied Biosystems). For primer sequences see Table S2.
Western blot analysis was performed on protein extracts from total trachea, epithelium and mesenchyme. Equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Membranes were blocked for 1 h with 5% (w/v) dried milk in PBS containing 0.1% Tween 20, and incubated with phospho-Smad1/5/8 antibody (1:1000; Cell Signaling, 9511), phospho-p38 antibody (1:2000; Cell Signaling, 4511), phospho-SAPK/JNK (Thr183/Tyr185) (G9) antibody (1:1000; Cell Signaling, 9255) and β-actin antibody (1:3000; Abcam, ab8226) in blocking buffer overnight at 4°C, followed by HRP-conjugated secondary antibody (Bio-Rad). Proteins were visualized using the ECL detection system (FEMTOMAX-110, Rockland Immunochemicals). The phospho-Smad1/5/8 band was validated by a positive control (HMEC1 cell treated with Bmp9) and phospho-p38 and phospho-Jun bands by molecular weight.

Protein extracts were collected from accessory lobes. Equal amounts of proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked for 1 h with 5% BSA in TBST (0.1% Tween 20) and then incubated with phospho-Smad1/5/8 antibody (Cell Signaling, 13820, 1:1000) and β-actin antibody (Abcam, ab8226, 1:2000) overnight at 4°C. Membranes were washed with TBST and then incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, 711-035-152, 715-035-151 1:10,000). Protein blots were analyzed with the ECL detection system (FEMTOMAX-110, Rockland Immunochemicals).