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ABLIM1 regulates VSMCs' contractile phenotype through interacting with MKL1. (A) Experimental flowchart of Co-immunoprecipitation assay combined with mass spectrometry. (B) Venn diagram showed the intersection of four proteins. (C) 293T cells were transfected with pcDNA3.1-Flag- Ablim1 and pcDNA3.1-His- Mkl1 . Lysates were immunoprecipitated with anti-Flag magnetic beads and blotted with anti-Flag <t>and</t> <t>anti-His</t> antibodies. (D) 293T cells were transfected with pcDNA3.1-Flag- Ablim1 and pcDNA3.1-His- Mkl1 . Lysates were immunoprecipitated with anti-His magnetic beads and blotted with anti-Flag and anti-His antibodies. (E) Molecular docking mode of ABLIM1 and MKL1. (F) Immunofluorescence images stained with anti-α-SMA, anti-MKL1 antibody in the VSMCs transfected with pcDNA3.1-Flag- Ablim1 and pcDNA3.1-Flag. (G) VSMCs were transfected with pcDNA3.1-Flag- Ablim1 and pcDNA3.1-His- Mkl1 , followed by TNFα treatment. Representative western blots and quantification of Flag, MKL1, MMP2, CNN1, α-SMA and SM22-α were shown. (H) VSMCs were transfected with si Ablim1 and si Mkl1 , followed by TNFα treatment. Representative western blots and quantification of ABLIM1, MKL1, MMP2, CNN1, α-SMA and SM22-α were shown. Data are presented as the mean ± SEM (n=3 per group). P-values were calculated by two-way ANOVA with Holm-Sidak multiple comparisons test. *** P<0.001 vs. pcDNA3.1-Flag + pcDNA3.1-His + TNFα or siControl + TNFα; ## P<0.01 vs. pcDNA3. 1-Flag- Ablim1 + pcDNA3.1-His + TNFα or si Ablim1 + TNFα, && P<0.01 vs. pcDNA3.1-Flag + pcDNA3.1-His- Mkl1 + TNFα or si Mkl1 + TNFα. ABLIM1, actin-binding LIM protein 1; VSMCs, vascular smooth muscle cells; VSMCs, vascular smooth muscle cells; MKL1, megakaryoblastic leukemia 1; α-SMA, α-smooth muscle actin; SM22α, smooth muscle 22α; MMP2, matrix metalloproteinase 2; CNN1, calponin 1; si-, small interfering.
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ABLIM1 regulates VSMCs' contractile phenotype through interacting with MKL1. (A) Experimental flowchart of Co-immunoprecipitation assay combined with mass spectrometry. (B) Venn diagram showed the intersection of four proteins. (C) 293T cells were transfected with pcDNA3.1-Flag- Ablim1 and pcDNA3.1-His- Mkl1 . Lysates were immunoprecipitated with anti-Flag magnetic beads and blotted with anti-Flag and anti-His antibodies. (D) 293T cells were transfected with pcDNA3.1-Flag- Ablim1 and pcDNA3.1-His- Mkl1 . Lysates were immunoprecipitated with anti-His magnetic beads and blotted with anti-Flag and anti-His antibodies. (E) Molecular docking mode of ABLIM1 and MKL1. (F) Immunofluorescence images stained with anti-α-SMA, anti-MKL1 antibody in the VSMCs transfected with pcDNA3.1-Flag- Ablim1 and pcDNA3.1-Flag. (G) VSMCs were transfected with pcDNA3.1-Flag- Ablim1 and pcDNA3.1-His- Mkl1 , followed by TNFα treatment. Representative western blots and quantification of Flag, MKL1, MMP2, CNN1, α-SMA and SM22-α were shown. (H) VSMCs were transfected with si Ablim1 and si Mkl1 , followed by TNFα treatment. Representative western blots and quantification of ABLIM1, MKL1, MMP2, CNN1, α-SMA and SM22-α were shown. Data are presented as the mean ± SEM (n=3 per group). P-values were calculated by two-way ANOVA with Holm-Sidak multiple comparisons test. *** P<0.001 vs. pcDNA3.1-Flag + pcDNA3.1-His + TNFα or siControl + TNFα; ## P<0.01 vs. pcDNA3. 1-Flag- Ablim1 + pcDNA3.1-His + TNFα or si Ablim1 + TNFα, && P<0.01 vs. pcDNA3.1-Flag + pcDNA3.1-His- Mkl1 + TNFα or si Mkl1 + TNFα. ABLIM1, actin-binding LIM protein 1; VSMCs, vascular smooth muscle cells; VSMCs, vascular smooth muscle cells; MKL1, megakaryoblastic leukemia 1; α-SMA, α-smooth muscle actin; SM22α, smooth muscle 22α; MMP2, matrix metalloproteinase 2; CNN1, calponin 1; si-, small interfering.

Journal: International Journal of Molecular Medicine

Article Title: miRNA-378a-5p attenuates the development of abdominal aortic aneurysm via ABLIM1-MKL1 signaling pathways

doi: 10.3892/ijmm.2026.5768

Figure Lengend Snippet: ABLIM1 regulates VSMCs' contractile phenotype through interacting with MKL1. (A) Experimental flowchart of Co-immunoprecipitation assay combined with mass spectrometry. (B) Venn diagram showed the intersection of four proteins. (C) 293T cells were transfected with pcDNA3.1-Flag- Ablim1 and pcDNA3.1-His- Mkl1 . Lysates were immunoprecipitated with anti-Flag magnetic beads and blotted with anti-Flag and anti-His antibodies. (D) 293T cells were transfected with pcDNA3.1-Flag- Ablim1 and pcDNA3.1-His- Mkl1 . Lysates were immunoprecipitated with anti-His magnetic beads and blotted with anti-Flag and anti-His antibodies. (E) Molecular docking mode of ABLIM1 and MKL1. (F) Immunofluorescence images stained with anti-α-SMA, anti-MKL1 antibody in the VSMCs transfected with pcDNA3.1-Flag- Ablim1 and pcDNA3.1-Flag. (G) VSMCs were transfected with pcDNA3.1-Flag- Ablim1 and pcDNA3.1-His- Mkl1 , followed by TNFα treatment. Representative western blots and quantification of Flag, MKL1, MMP2, CNN1, α-SMA and SM22-α were shown. (H) VSMCs were transfected with si Ablim1 and si Mkl1 , followed by TNFα treatment. Representative western blots and quantification of ABLIM1, MKL1, MMP2, CNN1, α-SMA and SM22-α were shown. Data are presented as the mean ± SEM (n=3 per group). P-values were calculated by two-way ANOVA with Holm-Sidak multiple comparisons test. *** P<0.001 vs. pcDNA3.1-Flag + pcDNA3.1-His + TNFα or siControl + TNFα; ## P<0.01 vs. pcDNA3. 1-Flag- Ablim1 + pcDNA3.1-His + TNFα or si Ablim1 + TNFα, && P<0.01 vs. pcDNA3.1-Flag + pcDNA3.1-His- Mkl1 + TNFα or si Mkl1 + TNFα. ABLIM1, actin-binding LIM protein 1; VSMCs, vascular smooth muscle cells; VSMCs, vascular smooth muscle cells; MKL1, megakaryoblastic leukemia 1; α-SMA, α-smooth muscle actin; SM22α, smooth muscle 22α; MMP2, matrix metalloproteinase 2; CNN1, calponin 1; si-, small interfering.

Article Snippet: The primary antibodies were as follows: anti-FLAG (1:1,000; cat. no. sc-166355; Santa Cruz Biotechnology, Inc.), anti-HIS (1:1,000; cat. no. 66005-1-Ig; Proteintech Group, Inc.), anti-ADM (1:1,000; cat. no. Ab190819 ; Abcam), anti-LMOD1 (1:1,000; cat. no. 15117-1-AP; Proteintech Group, Inc.) and anti-PRKCD (1:100; cat. no. sc-365969; Santa Cruz Biotechnology, Inc.).

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Transfection, Immunoprecipitation, Magnetic Beads, Immunofluorescence, Staining, Western Blot, Binding Assay