Journal: Development (Cambridge, England)

Article Title: The Gridlock transcriptional repressor impedes vertebrate heart regeneration by restricting expression of lysine methyltransferase

doi: 10.1242/dev.190678

Figure Lengend Snippet: Smyd2 functions as a KMT and controls Stat3 methylation and phosphorylation. (A) western blot analysis exhibiting Smyd2, Stat3, P-Stat3 or corresponding lysine methylated proteins using total lysates extracted from WT sibling, grl 5nt−/− , 4-HT-treated control Tg(cmlc2:nRSGG) hearts or 4-HT-treated Tg(cmlc2:creER;cmlc2:nRSGG) hearts at 7 dpa, respectively. GAPDH was used as a loading control. (B) Quantification of western blots using ImageJ software and normalized to GAPDH ( n =3). (C) Methylation and phosphorylation assay showing increased levels of methylated Stat3 and P-Stat3 in Flag-Stat3 and Myc-Smyd2a/HA-Smyd2b co-transfected HEK 293T cells. The cell lysates were used to immunoprecipitate (IP) Stat3 with anti-Flag antibody and then blotted with anti-methyl-lysine or anti-P-Stat3 antibodies. (D) Treatment with AZ505 or LLY-507 diminishes Stat3 methylation and phosphorylation in cells co-transfected with Flag-Stat3 and HA-Smyd2b. The cell lysates were treated with AZ505 (15 µM for 6 h, 25 µM for 6 h) or LLY-507 (2.5 µM for 28 h, 5 µM for 28 h), used to IP Stat3 using anti-Flag antibody and then blotted with anti-methyl-lysine and anti-P-Stat3 antibodies. (E) Quantification of methylated Stat3 and phosphorylated Stat3 levels normalized to Flag-Stat3 ( n =3). (F) Quantification of methylated Stat3 and phosphorylated Stat3 levels normalized to Flag-Stat3 in cells treated with AZ505 or LLY-507 ( n =3). (G,H) Smyd2a or Smyd2b immunoprecipitation with anti-Myc antibody (G) or anti-HA antibody (H), respectively, detects Stat3 using anti-Flag antibody. HEK 293T cells were co-transfected with constructs of Flag-Stat3 and Myc-Smyd2a (G) or HA-Smyd2b (H). GAPDH was used as a loading control. Data represents mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, Student's t -test (unpaired, two-tailed).

Article Snippet: 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).

Techniques: Methylation, Western Blot, Software, Phosphorylation Assay, Transfection, Immunoprecipitation, Construct, Two Tailed Test