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( A ) Illustration of the second position amino acid of AMOT130, AMOT130 ΔN , and rescue construct E2R_AMOT130 ΔN ; TNKS inhibition via XAV-939; and RNF146-mediated ubiquitylation of AMOT130. ( B ) Representative Western blot depicting AMOT130 ΔN and AMOT130 protein levels after TNKS inhibition by XAV-939 in primary skin fibroblasts derived from a female control, a male patient, or a male control. ( C ) Quantification of 5 independent Western blots shows that the protein levels of AMOT130 in male and female control primary skin fibroblasts are increased but AMOT130 ΔN level is not affected in patient primary skin fibroblasts upon XAV-939 treatment. The SD is reported. * P < 0.05, 2-way-ANOVA with Šídák’s multiple comparisons tests. ( D ) Representative Western blot depicting overexpressed AMOT130 ΔN and AMOT130 protein levels after XAV-939, JW 55, and <t>WIKI4</t> (10 μM, for 12 hours) addition to MCF7 cells. ( E ) Quantification of 5 independent Western blots shows that overexpressed AMOT130 protein level is increased, but overexpressed AMOT130 ΔN protein level is unaffected after XAV-939, JW 55, and WIKI4 treatment in MCF7 cells. The SD is reported. ** P ≤ 0.01, *** P < 0.001, 2-way ANOVA with Šídák’s multiple comparisons tests. ( F ) Representative Western blot demonstrating protein levels of AMOT130, AMOT130 ΔN , E2R_AMOT130 ΔN , and TBD_AMOT130 ΔN . E2R_AMOT130 ΔN mimics the destabilized N-terminus of AMOT130 by 1 amino acid substitution in the second position: glutamic acid to arginine (E2R) in AMOT130 ΔN . TBD_AMOT130 ΔN contains TBD 77–84 of wild-type AMOT130 at the N-terminus of AMOT130 ΔN . ( G ) Quantification of 5 independent Western blots shows that the protein level of overexpressed E2R_AMOT130 ΔN and TBD_AMOT130 ΔN is significantly decreased compared with AMOT130 ΔN but is still higher than AMOT130. The SD is reported. * P ≤ 0.05, ** P ≤ 0.01, 1-way ANOVA with Dunnett’s multiple comparisons test. (AMOT130 ΔN was taken as a control group.)
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( A ) Illustration of the second position amino acid of AMOT130, AMOT130 ΔN , and rescue construct E2R_AMOT130 ΔN ; TNKS inhibition via XAV-939; and RNF146-mediated ubiquitylation of AMOT130. ( B ) Representative Western blot depicting AMOT130 ΔN and AMOT130 protein levels after TNKS inhibition by XAV-939 in primary skin fibroblasts derived from a female control, a male patient, or a male control. ( C ) Quantification of 5 independent Western blots shows that the protein levels of AMOT130 in male and female control primary skin fibroblasts are increased but AMOT130 ΔN level is not affected in patient primary skin fibroblasts upon XAV-939 treatment. The SD is reported. * P < 0.05, 2-way-ANOVA with Šídák’s multiple comparisons tests. ( D ) Representative Western blot depicting overexpressed AMOT130 ΔN and AMOT130 protein levels after XAV-939, JW 55, and WIKI4 (10 μM, for 12 hours) addition to MCF7 cells. ( E ) Quantification of 5 independent Western blots shows that overexpressed AMOT130 protein level is increased, but overexpressed AMOT130 ΔN protein level is unaffected after XAV-939, JW 55, and WIKI4 treatment in MCF7 cells. The SD is reported. ** P ≤ 0.01, *** P < 0.001, 2-way ANOVA with Šídák’s multiple comparisons tests. ( F ) Representative Western blot demonstrating protein levels of AMOT130, AMOT130 ΔN , E2R_AMOT130 ΔN , and TBD_AMOT130 ΔN . E2R_AMOT130 ΔN mimics the destabilized N-terminus of AMOT130 by 1 amino acid substitution in the second position: glutamic acid to arginine (E2R) in AMOT130 ΔN . TBD_AMOT130 ΔN contains TBD 77–84 of wild-type AMOT130 at the N-terminus of AMOT130 ΔN . ( G ) Quantification of 5 independent Western blots shows that the protein level of overexpressed E2R_AMOT130 ΔN and TBD_AMOT130 ΔN is significantly decreased compared with AMOT130 ΔN but is still higher than AMOT130. The SD is reported. * P ≤ 0.05, ** P ≤ 0.01, 1-way ANOVA with Dunnett’s multiple comparisons test. (AMOT130 ΔN was taken as a control group.)

Journal: The Journal of Clinical Investigation

Article Title: A pathogenic variant of AMOT leads to isolated X-linked congenital hydrocephalus due to N-terminal truncation

doi: 10.1172/JCI179438

Figure Lengend Snippet: ( A ) Illustration of the second position amino acid of AMOT130, AMOT130 ΔN , and rescue construct E2R_AMOT130 ΔN ; TNKS inhibition via XAV-939; and RNF146-mediated ubiquitylation of AMOT130. ( B ) Representative Western blot depicting AMOT130 ΔN and AMOT130 protein levels after TNKS inhibition by XAV-939 in primary skin fibroblasts derived from a female control, a male patient, or a male control. ( C ) Quantification of 5 independent Western blots shows that the protein levels of AMOT130 in male and female control primary skin fibroblasts are increased but AMOT130 ΔN level is not affected in patient primary skin fibroblasts upon XAV-939 treatment. The SD is reported. * P < 0.05, 2-way-ANOVA with Šídák’s multiple comparisons tests. ( D ) Representative Western blot depicting overexpressed AMOT130 ΔN and AMOT130 protein levels after XAV-939, JW 55, and WIKI4 (10 μM, for 12 hours) addition to MCF7 cells. ( E ) Quantification of 5 independent Western blots shows that overexpressed AMOT130 protein level is increased, but overexpressed AMOT130 ΔN protein level is unaffected after XAV-939, JW 55, and WIKI4 treatment in MCF7 cells. The SD is reported. ** P ≤ 0.01, *** P < 0.001, 2-way ANOVA with Šídák’s multiple comparisons tests. ( F ) Representative Western blot demonstrating protein levels of AMOT130, AMOT130 ΔN , E2R_AMOT130 ΔN , and TBD_AMOT130 ΔN . E2R_AMOT130 ΔN mimics the destabilized N-terminus of AMOT130 by 1 amino acid substitution in the second position: glutamic acid to arginine (E2R) in AMOT130 ΔN . TBD_AMOT130 ΔN contains TBD 77–84 of wild-type AMOT130 at the N-terminus of AMOT130 ΔN . ( G ) Quantification of 5 independent Western blots shows that the protein level of overexpressed E2R_AMOT130 ΔN and TBD_AMOT130 ΔN is significantly decreased compared with AMOT130 ΔN but is still higher than AMOT130. The SD is reported. * P ≤ 0.05, ** P ≤ 0.01, 1-way ANOVA with Dunnett’s multiple comparisons test. (AMOT130 ΔN was taken as a control group.)

Article Snippet: MCF7 cells were seeded at the density of 2 × 10 –5 /well in 12-well plates for (a) AMOT130, AMOT130 ΔN , and chimeric protein overexpression; (b) XAV-939 (10 μM; Selleckchem), JW 55, and WIKI4 (10 μM; MedChemExpress) treatment; (c) BMP6 stimulation; and (d) IF staining (cells seeded on coverslips) experiments.

Techniques: Construct, Inhibition, Western Blot, Derivative Assay, Control