Journal: Neurology: Genetics

Article Title: Novel Cysteine-Sparing Hypomorphic NOTCH3 A1604T Mutation Observed in a Family With Migraine and White Matter Lesions

doi: 10.1212/NXG.0000000000000584

Figure Lengend Snippet: (A) Western blot analysis of the inactivation of NOTCH3 by CRISPR/Cas9 in the 293T(ΔN1-N3) cells (B). Western blot analysis showing the levels of full-length wild-type and A1604T NOTCH3 receptor in the 293T(ΔN1-N3)N3wt (wt) and 293T(ΔN1-N3)N3A1604T (A1604T) cell lines, respectively, compared with 293T(ΔN1-N3) cells as the control (left). β-Actin levels were used as loading controls. The positions of the FL form of the receptor are indicated. On the right: quantification of the full-length receptor in the 293T(ΔN1-N3)N3wt and 293T(ΔN1-N3)N3A1604T cell lines. (C). Analysis of messenger RNA (mRNA) expression from the HES5, HEY1 , and NRARP genes in the 293T(ΔN1-N3)N3wt and 293T(ΔN1-N3)N3A1604T cell lines or in 293T cells as the control in response to activation of the wild-type or A1604T NOTCH3 receptor by Jagged1 (Notch on) or block of S3 cleavage by DAPT treatment (Notch off), as indicated. β-Actin levels were used as loading controls, and the positions of the FL form of the receptor are indicated. GAPDH mRNA expression was used for normalization. Values are significant at *** p < 0.001, ** p < 0.01, and * p < 0.05. FL = full length.

Article Snippet: To block Notch signaling, cells were treated with 10 μM of the γ-secretase inhibitor DAPT (Merck Millipore, Darmstadt, Germany) for 24 hours.

Techniques: Western Blot, CRISPR, Control, Expressing, Activation Assay, Blocking Assay

Journal: Neurology: Genetics

Article Title: Novel Cysteine-Sparing Hypomorphic NOTCH3 A1604T Mutation Observed in a Family With Migraine and White Matter Lesions

doi: 10.1212/NXG.0000000000000584

Figure Lengend Snippet: (A) Schematic presentation of Notch receptor processing (S1, S2, and S3 cleavage and the ECD, TMIC, NEXT, and ICD moieties). The locations of the NRR the HD (in blue) and the Lin12/Notch repeats (LNR) are also depicted. (B). Western blot analysis of TMIC levels in the 293T(ΔN1-N3)N3wt (wt) and 293T(ΔN1-N3)N3A1604T (A1604T) cell lines (left) with quantification of the TMIC levels to the right. Note that the β-actin Western blot in is the same as in , i.e., the full-length NOTCH3 band in and the TMIC band in are derived from the same Western blot and thus share the same β-actin loading control. (C). Western blot analysis of TMIC and NEXT/ICD fragments from the 293T(ΔN1-N3)N3wt and 293T(ΔN1- N3)N3A1604T cell lines, following activation by ligand (Jagged1) or blockade of S3 cleavage (DAPT), as indicated. The signal intensity (as TMIC, NEXT-ICD, ratio TMIC/NEXT-ICD, and β-actin) for the Western blot is shown below. (D). Western blot analysis of biotin cell surface labeling of the NOTCH3 receptor, using an antibody to the NOTCH3 ICD (left). The protein amounts from input (input) and following immunoprecipitation for biotin (IP: Biotin) are shown for the 293T(ΔN1-N3)N3wt (wt) and 293T(ΔN1-N3)N3A1604T (A1604T) cell lines. To the right, the ratio of TMIC immunoprecipitated over TMIC input is shown. β-Actin levels were used as loading controls, and the positions of the full-length (FL), TMIC, and NEXT/ICD forms of the receptor are indicated. Values are significant at *** p < 0.001 and * p < 0.05. ECD = extracellular domain; HD = heterodimerization domain; ICD = intracellular domain; NRR = negative regulatory region; TMIC = transmembrane and intracellular.

Article Snippet: To block Notch signaling, cells were treated with 10 μM of the γ-secretase inhibitor DAPT (Merck Millipore, Darmstadt, Germany) for 24 hours.

Techniques: Western Blot, Derivative Assay, Control, Activation Assay, Labeling, Immunoprecipitation

Journal: Biology Open

Article Title: Jagged1 protein processing in the developing mammalian lens

doi: 10.1242/bio.041095

Figure Lengend Snippet: DAPT-treatment of human lens B3 cells induces changes in endogenous JAG1 protein processing. (A) Endogenous JAG1, NOTCH2 and PSEN1 expression in B3 cell lysates. Each lane/strip blot contains 50 µg of B3 cell total protein that was separately probed with rabbit anti-Jag1, rabbit anti-Psen1 (N-terminus), or rat anti-Notch2 to reveal expression of FL-JAG1, PSEN1 and NOTCH2 proteins. The JAG1-CTF isoform was observable in longer exposures (not shown, see control lanes in Fig. 3 B,C). The NOTCH2 full length and N2-ICD isoforms were clearly detectable. (B–C) B3 cells treated with different concentrations of Epoxomicin versus DMSO (B), or 10 µM DAPT versus DMSO (C) for 24 h and cell lysates analyzed for JAG1 protein isoforms via western blot using rabbit anti-Jag1 antibody. (D) Western blotting of B3 cells transfected with C-terminal HA-tagged isoforms of rat Jag1 protein (50 µg total protein loaded per lane; blot probed with rabbit anti-Jag1 antibody). (E) B3 cells transfected with either FL-Jag1-HA of Jag1-CTF-HA constructs, followed by treatment with 10 µM DAPT (versus DMSO alone) for 24 h. Western blot probed with rabbit anti-Jag1. In all panels, blots were reprobed with mouse anti β-Actin as a loading control. Each blot is representative of n =3 independent biological replicates.

Article Snippet: At 24 h post-transfection, cells were treated with the γ-secretase inhibitor DAPT (APExBIO, Cat#:A8200) at a concentration of 10 μM in DMSO, or DMSO alone in controls, for an additional 24 h before being harvested as stated above.

Techniques: Expressing, Stripping Membranes, Control, Western Blot, Transfection, Construct

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Microarray Analysis of Long Non-Coding RNAs in Lung Tissues of Patients with COPD and HOXA-AS2 Promotes HPMECs Proliferation via Notch1

doi: 10.2147/COPD.S259601

Figure Lengend Snippet: HOXA-AS2 promote HPMECs proliferation through regulating Notch1. ( A ) HOXA-AS2 siRNAs were transfected into HPMECs for 24 h, and the relative expression levels of Notch1 were measured by real-time PCR. Notch1 mRNA decreased in HOXA-AS2 knocking down HPMECs. ( B – D ) Western blot was used to analyze the expression of Notch1 protein after transfection with HOXA-AS2 siRNA, overexpressing HOXA-AS2 lentivirus, Notch1 overexpression plasmid or Notch1 inhibitor gamma-Secretase Inhibitor IX (GSI-IX, DAPT 10 mM for 24 h) in HPMECs. ( E – G ) Cell Counting Kit-8 (CCK-8) assay was used to measure HPMECs viability after treatment. All data are presented as mean ± SEM. * P < 0.05. All experiments were performed independently at least 3 times. Abbreviations: HPMECs, human pulmonary microvascular endothelial cells; real-time-PCR, real-time polymerase chain reaction; CCK-8, Cell Counting Kit-8.

Article Snippet: The cells were seeded into 96 wells, and treated with the HOXA-AS2 siRNA, or CSE at different concentrations of 0, 0.5%, 1%, 2.5%, and 5%, or 10 mM of Notch1 inhibitor DAPT (GSI-IX, gamma-Secretase Inhibitor IX, ApexBio Technology, TX, USA) for 24h.

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Plasmid Preparation, Cell Counting, CCK-8 Assay