Journal: Nature Communications

Article Title: NOTCH1 mitochondria localization during heart development promotes mitochondrial metabolism and the endothelial-to-mesenchymal transition in mice

doi: 10.1038/s41467-024-54407-7

Figure Lengend Snippet: A Representative western blot illustrating the intracellular distribution of pyruvate dehydrogenase complex (PDC) components in wild-type and NOTCH1 knockout ( NOTCH1 KO) human embryonic kidney 293T (HEK293T) cells. PDHB pyruvate dehydrogenase E1 subunit beta, DLAT dihydrolipoamide S-acetyltransferase; DLD dihydrolipoyl transacetylase. B Endogenous co-immunoprecipitation (CO-IP) of NICD1 with PDC components in human umbilical vein endothelial cells (HUVEC) and HEK293T cells. C GST pull-down assay to determine in vitro binding of NICD1 to PDHA1, PDHB, DLD, and DLAT. D Representative western blot illustrating the level of each PDC component collected by endogenous immune-precipitation (IP) from wild-type HEK293T cells, NOTCH1 KO HEK293T cells, NOTCH1 KO HEK293T cells expressing exogenous NICD1, or recombinant mitochondria-targeted NICD1 (mitoNICD1). E Representative western blot illustrating the level of p-PDHA1 at serine 293 (Ser293), serine 232 (Ser232), and serine 300 (Ser300) in HUVEC cells expressing NICD1 or mitoNICD1 (left) and in HEK293T cells expressing NICD1 or mitoNICD1 (right). F Representative western blot illustrating the level of p-PDHA1 at Ser293, Ser232, and Ser300 in wild type and NOTCH1 knockdown ( NOTCH1 KD) HUVEC cells (left), and wild-type and NOTCH1 KO HEK293T cells (right). G Representative western blot illustrating the affinity of PDKs, including PDK1, PDK2, PDK3, and PDK4, and pyruvate dehydrogenase phosphatases (PDPs), including PDP1 and PDP2, to endogenous PDHA1 collected by endogenous IP from HUVEC cells expressing NICD1, mitoNICD1 or not (left), and HEK293T cells expressing NICD1, mitoNICD1, or not (right). H Representative western blot illustrating the affinity of PDK1 and PDP1 to endogenous PDHA1 collected by endogenous IP from wild-type mice and Notch1 heterozygous knockout ( Notch1 +/− ) mice. I Representative western blot illustrating the p-PDHA1 level at Ser293, Ser232, and Ser300 in mouse cardiac endothelial cells isolated from wild-type and Notch1 +/− mice. Three experiments were repeated independently with similar results for A – I . Source data are provided as a Source Data file Source data are provided as a Source Data file.

Article Snippet: After blocking with 5% skim milk, membranes were incubated with specific primary antibodies, including NOTCH1 (1:1000, #3608, CST, MA, USA), Histone H3 (H3) (1:5000, P30266, abmart, Shanghai, China), GAPDH (1:5000, 30201ES20, Yeasen, Shanghai, China), COX4 (1:3000, #4850, CST), FLAG (1:5,000, M20008, abmart), HA (1:5,000, M20021, abmart), CS (1:1,000, EPR8067, Abcam), ACTIN (1:5000, 30102ES40, Yeasen), pyruvate dehydrogenase E1 subunit alpha (PDHA1) (1:1,000, #3205, CST), PDHB (1:1000, 14744-1-AP, Proteintech, IL, USA), dihydrolipoyl dehydrogenase (DLD) (1:1000, 16431-1-AP, Proteintech), dihydrolipoamide S-acetyltransferase (DLAT) (1:1000, #12362, CST), pyruvate dehydrogenase kinase (PDK) 1 (PDK1) (1:1000, #3820, CST), PDK2 (1:1000, 15647-1-AP, Proteintech), PDK3 (1:1000, 12215-1-AP, Proteintech), PDK4 (1:1000, 12949-1-AP, Proteintech), p-PDHA (1:500, S293) (ab177461, Abcam), p-PDHA (S232) (1:500, 29582-1-AP, Proteintech), p-PDHA (S300) (1:500, 80572-1-RR, Proteintech), CD31 (1:1000, #3528, CST), VE-Cadherin (1:1000, VE-Cad, #3528, CST), vascular endothelial growth factor receptor (VEGFR) (1:1000, #2893, CST), α smooth muscle actin (1:1000,α-SMA) (14395-1-AP, Proteintech), N-Cadherin (1:500, N-Cad, #4061, CST), and Vimentin (1:500, 10366-1-AP, Proteintech), overnight at 4 °C.

Techniques: Western Blot, Knock-Out, Immunoprecipitation, Co-Immunoprecipitation Assay, Pull Down Assay, In Vitro, Binding Assay, Expressing, Recombinant, Knockdown, Isolation