The supernatant was transferred into a new tube and stored at -80 °C. Conditioned media (2 ml each) were concentrated into 100 μL using 3 kD MWCO spin column and 30 μL was mixed with 4× Laemmli buffer, and proteins were separated by 4-12% Bis-Tris gel at 130 V for 1.5 h. The gels were silver stained; the darker bands were observed in stretched LRP6-overexpressed CMs, and compared with the control group. The chosen bands were cut and digested overnight in the digestion solution (trypsin 12.5 ng/μL and 20 mM NH4HCO3). The digested peptides were lyophilized and re-suspended with 32μl solvent C (C: water with 0.1% formic acid; D: ACN with 0.1% formic acid), separated by nanoLC and analyzed by online electrospray tandem mass spectrometry. The experiment was performed on a Nano Aquity UPLC system (Waters Corporation, Milford, MA), which was connected to a quadrupole-Orbitrap mass spectrometer (Q-Exactive) (Thermo Fisher Scientific, Bremen, Germany) equipped with an online nano-electrospray ion source. The peptide sample (4 μl) was loaded onto the trap column (Thermo Scientific Acclaim PepMap C18, 100 μm × 2 cm) and subsequently separated on the analytical column (Acclaim PepMap C18, 75 μm × 25 cm) with a linear gradient from 5% D to 30% D in 85 min. The column was re-equilibrated at initial conditions for 5 min. The column flow rate was maintained at 300 nL/min, and the column temperature was maintained at 45 °C. The electrospray voltage of 2.0 kV versus the inlet of the mass spectrometer was used.

All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.3) set up to search the NCBI Rattus norvegicus database assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.050 Da and a parent ion tolerance of 10.0 ppm. Carbamidomethyl of cysteine was specified in Mascot as fixed modifications. Oxidation of methionine was specified in Mascot as a variable modification.

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