Journal: Molecules and Cells

Article Title: Integrated Quantitative Phosphoproteomics and Cell-Based Functional Screening Reveals Specific Pathological Cardiac Hypertrophy-Related Phosphorylation Sites

doi: 10.14348/molcells.2021.4002

Figure Lengend Snippet: (A) Overall scheme of disease-specific phosphorylated site selection. Only 47 phosphoproteins among 134 upregulated phosphoproteins were identified as phosphoproteins related to heart and muscle disease in the human disease database. Twenty-nine phosphoproteins were mapped in the heart tissue-specific expression database and literature. The changes at each phosphorylation site were compared with its protein abundance to identify phosphorylation-specific level changes. Finally, 14 target phosphoproteins and phosphorylation competition peptides for 18 phosphorylation target sites were selected for cell-based screening. (B) Cultured neonatal rat cardiomyocytes were treated with 100 nM competition peptides to inhibit site-specific phosphorylation and exposed to 100 µM PE for 24 h. The cell surface area was then determined by immunofluorescence staining of α-actinin, and bar graphs representing surface area were drawn. Statistical significance was assessed by two-tailed Student’s t -tests for each comparison group with three biological replicates. (C) Immunofluorescence images of sarcomeric α-actinin (a, untreated; b, PE; c, TAT; d, TAT + PE; e, Ldb3-S98 + PE; f, Ldb3-S121, 123 + PE; g, Ldb3-S170, 171 + PE; h, Ldb3-S179 + PE; i, Palld-S901 + PE; j, Pallad-S1146 + PE). PE, phenylephrine; TAT, transactivator of transcription. (D) Bar graphs showing changes in cell surface area following treatment with different concentrations of competition peptides targeting Ser-98 and Ser-179 of Ldb3. *** P < 0.001 vs untreated; ## P < 0.01, ### P < 0.001 vs TAT + PE; n.s., not significant; n = 3 per group.

Article Snippet: The antibodies and dilutions used for immunoblotting were as follows: 1:10,000 phospho-Ser-98 and phospho-Ser-179 Ldb3 polyclonal antibodies (AbFrontier, Korea); 1:5,000 Cypher (sc-136380; Santa Cruz Biotechnology, USA); and 1:2,500 GAPDH (sc-32233; Santa Cruz Biotechnology).

Techniques: Selection, Expressing, Cell Culture, Immunofluorescence, Staining, Two Tailed Test

Journal: Molecules and Cells

Article Title: Integrated Quantitative Phosphoproteomics and Cell-Based Functional Screening Reveals Specific Pathological Cardiac Hypertrophy-Related Phosphorylation Sites

doi: 10.14348/molcells.2021.4002

Figure Lengend Snippet: Heart lysates were immunoblotted with non-phospho-antibody, Ser-98 site-specific phospho-antibody, and Ser-179 site-specific phospho-antibody in (A) 8-week-old and (B) 12-week-old JTT-1 TG mice. Bar charts show the relative intensity of site-specific phosphorylation compared with wild-type expression. P values determined using two-tailed Student’s t -tests; * P < 0.05, ** P < 0.01; n.s., not significant; n = 3 per group. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control.

Article Snippet: The antibodies and dilutions used for immunoblotting were as follows: 1:10,000 phospho-Ser-98 and phospho-Ser-179 Ldb3 polyclonal antibodies (AbFrontier, Korea); 1:5,000 Cypher (sc-136380; Santa Cruz Biotechnology, USA); and 1:2,500 GAPDH (sc-32233; Santa Cruz Biotechnology).

Techniques: Expressing, Two Tailed Test

Journal: Molecules and Cells

Article Title: Integrated Quantitative Phosphoproteomics and Cell-Based Functional Screening Reveals Specific Pathological Cardiac Hypertrophy-Related Phosphorylation Sites

doi: 10.14348/molcells.2021.4002

Figure Lengend Snippet: Changes in site-specific phosphorylation of Ser-98 and Ser-179 of Ldb3 were investigated in (A) physiological cardiac hypertrophy (swimming training for 2 or 4 weeks), and (B) pathological cardiac hypertrophy (TAC and sham surgery for 2 weeks). Lysates were probed with non-phospho- and phospho-antibodies (recognizing Ser-98 or Ser-179 of Ldb3) to detect changes in endogenous site-specific phosphorylation. * P < 0.05, ** P < 0.01; n.s., not significant; n = 3 per group. con, control; sw, swimming.

Article Snippet: The antibodies and dilutions used for immunoblotting were as follows: 1:10,000 phospho-Ser-98 and phospho-Ser-179 Ldb3 polyclonal antibodies (AbFrontier, Korea); 1:5,000 Cypher (sc-136380; Santa Cruz Biotechnology, USA); and 1:2,500 GAPDH (sc-32233; Santa Cruz Biotechnology).

Techniques: