Anti smyd2

Ventricles from 4-HT-treated Tg(cmlc2:creER;cmlc2:nRSGG) and Tg(cmlc2:nRSGG) animals, as well as grl^5nt−/− and WT sibling zebrafish at 7 dpa were collected and homogenized with an electric homogenizer in cold RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100 and 0.1% SDS) supplemented with proteinase inhibitors (Millipore) and phosphatase inhibitor cocktail (Apex Bio). The lysates were loaded into 12.5% SDS-PAGE after boiling for 5 min in SDS-PAGE sample loading buffer. Proteins were then transferred to PVDF membranes using a Trans-Blot Turbo Transfer System (Bio-Rad). Signals were detected by the Clarity Western ECL Substrate (Bio-Rad) and scanned using a ChemiScope series system (Clinx). Antibodies used for western blot in this study were as follows: anti-Smyd2 (Cell Signaling; #9734; 1:500), anti-P-Stat3 (Santa Cruz; sc-8059; 1:500), anti-Stat3 (Santa Cruz; sc-8019; 1:500), anti-methyl-lysine (Abbkine; ABM0060; 1:1000), anti-GAPDH (Abcam; ab181602; 1:10,000), anti-GAPDH (Abmart; M20006L; 1:4000), anti-actin (Sigma; A5441; 1:4000), anti-Flag (Sigma; 7425/1804; 1:4000), anti-Myc (HuaAn Biotechnology; R1208-1; 1:4000), anti-HA (Santa Cruz; sc-805; 1:4000), HRP-anti-rabbit IgG (Cwbio; CW0103S; 1:5000) and HRP-anti-mouse IgG (Cwbio; CW0102S; 1:5000).

An anti-K133 monomethylated β-catenin antibody was generated in rabbit by immunization with a synthetic peptide. The amino acid sequences of modified and unmodified peptides are shown in Supplementary Table 2. The following primary antibodies were used for western blot (WB) and immunocytochemical (ICC) analysis: anti-K133 monomethylated β-catenin antibody (1:500 dilution for WB and 1:200 dilution for ICC); anti-FLAG tag (M2, mouse, #F3165; Sigma-Aldrich, St. Louis, MO; 1:2000 dilution for WB and 1:500 dilution for ICC), anti-SMYD2 (D14H7, rabbit, #9734; Cell Signaling Technology; 1:500 dilution for WB and 1:100 dilution for ICC), anti-β-catenin (carboxy-terminal antigen, rabbit, #9587; Cell Signaling Technology; 1:500 dilution for WB and 1:100 dilution for ICC), anti-α-tubulin (DM1A, mouse; CALBIOCHEM, Billerica, MA; 1:500 dilution for WB and 1:100 dilution for ICC), anti-histone H3 (rabbit, ab1791; Abcam; 1:500 dilution for WB), anti-HA (3F10, rat, #11867423001; Roche;1:2000 dilution for WB and 1:500 dilution for ICC ), anti-FOXM1 (D12D5, Rabbit, #5436S; Cell Signaling Technology; 1:500 dilution for WB and 1:100 dilution for ICC), anti-BCL9 (rabbit, #15096S, Cell Signaling Technology, 1:500 dilution for WB).