Pab15842

Cells were harvested and homogenized with RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, and protease inhibitor cocktail). Protein was denatured in sodium dodecyl sulfate (SDS) buffer and separated with SDS-PAGE gels. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and then blocked with 5% skim milk (OXOID). Membranes were incubated with primary antibody overnight at 4, and then incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG antibody (1:10000, Novex, Carlsbad, CA) at room temperature for 1 hour. Visualization was performed with WesternBright ECL HRP (Bio-Rad). β-actin (1:500, Santa Cruz) was used as the internal control.
The same procedure was used to determine the IFT80 (1:400, PAB15842, Abnova), FGFR1 (1:1000, ab10646, Abcam), p-FGFR1 (1:1000, ab59194, Abcam), Smad1/5/8 (1:300, sc-6031-R, Santa Cruz), p-Smad1/5/8 (1:300, sc-12353-R, Santa Cruz), AKT (1:300, sc-8312, Santa Cruz), p-AKT (1:300, sc-7985-r, Santa Cruz).

Total RNA from D14 fractured calluses and POBs were isolated with Trizol Reagent (Life Technologies) and cDNA was prepared from 1 μg total RNA using the PrimeScriptTM RT reagent kit (Takara Bio). Quantitative real-time RT-PCR was performed with SYBR Green PCR master Mix (Invitrogen). The relative gene expression was calculated as described previously [9 (link)]. Primer sequences are listed in Table 2. Western blots from D21 fractured callus samples were carried out as previously described [9 (link), 18 (link)] using rabbit anti-IFT80 antibody (1:400, PAB15842, Abnova) and rabbit anti-Foxo1 antibody (1:500, 2880S, Cell Signaling). Beta-actin was used as a loading control. Protein band intensities were measured using ImageJ software and normalized to beta-actin.Table 2

List of RT-qPCR primer sequences.

Name	Forward primer sequence	Reverse primer sequence
IFT80	AAGGAACCAAAGCATCAAGAATTAG	AGATGTCATCAGGCAGCTTGAC
OSX	AGCGACCACTTGAGCAAACAT	GCGGCTGATTGGCTTCTTCT
ALP	ATCTTTGGTCTGGCTCCCATG	TTTCCCGTTCACCGTCCAC
Col-1	GCAACAGTCGCTTCACCTACA	CAATGTCCAAGGGAGCCACAT
Foxo1	GCTGCATCCATGGACAACAACA	CGAGGGCGAAATGTACTCCAGTT
GAPDH	ACTTTGTCAAGCTCATTTCC	TGCAGCGAACTTTATTGATG

Cells were harvested and homogenized with radioimmunoprecipitation assay buffer. Proteins were denatured in sodium dodecyl sulfate (SDS) buffer and separated with SDS-polyacrylamide gel electrophoresis gels. Proteins were transferred to polyvinylidene difluoride membranes and then blocked with 5% skimmed milk (OXOID). Membranes were incubated with primary antibody overnight at 4 °C, and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (1:10,000, Novex, Carlsbad, CA, USA) at room temperature for 1 h. Visualization was performed with WesternBright ECL HRP (Bio-Rad). β-Actin (1:500, Santa Cruz) was used as the internal control.
The same procedure was used to determine the IFT80 (1:400, PAB15842, Abnova), AKT (1:300, sc-8312, Santa Cruz), and p-AKT (1:300, sc-7985-r, Santa Cruz).