Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Deficiency of FABP7 Triggers Premature Neural Differentiation in Idiopathic Normocephalic Autism Organoids.

doi: 10.1002/advs.202406849

Figure Lengend Snippet: Figure 6. MEK2 overexpression causes autistic-like behaviors in mice, and its inhibition can rescue premature differentiation in ASD organoids. A) Schematic of AAV2/9 vectors for expressing MEK2-EGFP. B) Immunostaining images displaying GFP expressed in the hippocampus, including the dentate gyrus (DG) and CA, colabeled with the DG granule cell marker Prox1. Scale bar: 200 μm (left), 50 μm (right). C) Schematic of the open field test (Left) and histograms (Right) presenting the center zoneduration time (AAV-NC: n = 14 mice, AAV-MEK2: n = 15 mice, ns p = 0.86) and center zone entries (AAV-NC: n = 14 mice, AAV-MEK2: n = 15 mice, ns p = 0.31) of the mice in the AAV-NC and AAV-MEK2 groups. D) Schematic of the 3-chambered

Article Snippet: CTIP2 Rat IgG 1:500(IF) abcam ab18465 DCX Rabbit IgG 1:1000(IF) Cell Signaling 4604 FABP7 Rabbit IgG 1:2000(WB) 1:500(IP) Cell Signaling Technology 13347S FOXG1 Rabbit IgG 1:1000(IF) abcam ab18259 FOXP2 Rabbit IgG 1:1000(IF) abcam ab16046 GAPDH Mouse IgG 1:5000(WB) affinity T0004 GFP Chicken IgG 1:1000(IF) Millipore ab16901 GFP Rabbit IgG 1:1000(IF) Chemicon AB3080 KI67 Rabbit IgG 1:500(IF) Invitrogen 180191Z MAP4K2 Rabbit IgG 1:2000(WB) abcam ab184169 MEK1/2 Mouse IgG 1:1000(WB) Cell Signaling Technology 4694S NANOG Goat IgG 1:1000(IF) R&D Systems AF1997 NESTIN Goat IgG 1:1000(IF) Santa Cruz SC-21247 PAX6 Rabbit IgG 1:500(IF) convance PRB-278P PHH3 Rat IgG 1:1000(IF) Cell Signaling Technology 9706S PKCλ Mouse IgG 1:1000(IF) BD 610 207 p-MEK1/2 (S217/221) Rabbit IgG 1:1000(WB) Cell Signaling Technology 3958S PROX1 Rabbit IgG 1:300(IF) abcam ab101851 p-Vimentin Mouse IgG 1:1000(IF) MBL D076-3 SOX2 Goat IgG 1:500(IF) R&D Systems AF2018 TBR1 Rabbit IgG 1:500(IF) abcam ab31940 γ-tublin Mouse IgG 1:1000(IF) abcam ab11316 Alexa Fluor 488 Goat anti-Chicken IgG 1:1000(IF) Invitrogen A11039 Alexa Fluor 488 Donkey anti-Rat IgG 1:1000(IF) Invitrogen A21208 Alexa Fluor 488 Donkey anti-Rabbit IgG 1:1000(IF) Invitrogen A21206 Alexa Fluor 488 Donkey anti-Mouse IgG 1:1000(IF) Invitrogen A21202 Alexa Fluor 488 Donkey anti-Goat IgG 1:1000(IF) Invitrogen A11055 Alexa Fluor 546 Donkey anti-Rabbit IgG 1:1000(IF) Invitrogen A10040 Alexa Fluor 546 Donkey anti-Mouse IgG 1:1000(IF) Invitrogen A10036 Alexa Fluor 546 Goat anti-Rat IgG 1:1000(IF) Invitrogen A11081 Alexa Fluor 546 Donkey anti-Goat IgG 1:1000(IF) Invitrogen A11056 Alexa Fluor 647 Donkey anti-Mouse IgG 1:1000(IF) Invitrogen A31571 Alexa Fluor 647 Donkey anti-Rabbit IgG 1:1000(IF) Invitrogen A31573 Alexa Fluor 647 Donkey anti-Goat IgG 1:1000(IF) Invitrogen A21447 Hoechst332258 1:1000(IF) Invitrogen A1339 HRP Goat Anti-Mouse IgG 1:5000(WB) Multi Sciences 70-GAM0072 HRP Goat Anti-Rabbit IgG 1:5000(WB) Multi Sciences 70-GAR0072 was reverse transcribed into cDNA using a PrimeScriptTM RT reagent kit (TaKaRa) and prepared for qPCR using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific).The primers for qPCR used were as follows: FABP7 forward primer, 5′-TCTCACCTCCTTCCTTCTTCT-3′ and reverse primer, 5′-AGAACCTTG CCAGTGATGTATT-3′; MEK2 forward primer, 5′-CTCACCATCAACCCTAC CATC-3′, and reverse primer, 5′-GCAGGTCCACCAGGTTT-3′; GAPDH forward primer, 5′-TCGACAGTCAGCCGCATCTTCTTT-3′ and reverse primer, 5′-ACCAAATCCGTTGACTCCGACCTT-3′.

Techniques: Over Expression, Inhibition, Expressing, Immunostaining, Marker

Journal: Cardiovascular research

Article Title: Molecular mechanisms of activation of endothelial nitric oxide synthase mediated by transient receptor potential vanilloid type 1.

doi: 10.1093/cvr/cvr104

Figure Lengend Snippet: Figure 2 Ca2+-dependent PI3K/Akt/CaMKII mediates TRPV1 ligand-induced eNOS activation and NO production in BAECs. (A) BAECs were treated with 1 mmol/L evodiamine for the indicated times. (B–D) BAECs were pretreated with or without 10 mmol/L LY294002, 5 mmol/L KN62, or 500 nmol/L EGTA for 1 h, then incubated with evodiamine (1 mmol/L) for 15 min. (E) Cells were incubated with DMSO, LY294002 (10 mmol/L), KN62 (5 mmol/L), or EGTA (500 nmol/L) for 1 h, then evodiamine (1 mmol/L) for 24 h. (F) HAECs were transfected with scramble (200 nmol/L) or TRPV1 siRNA (200 nmol/L) for 24 h, then incubated with evodiamine (1 mmol/L) for an additional 15 min. Western blot analysis involved the use of antibodies against phosphorylated Akt at Ser473, Akt protein, phosphorylated CaMKII at Thr286, CaMKII protein, phosphorylated eNOS at Ser1179, and eNOS protein. Level of nitrite in medium was analysed by Griess assay. *P , 0.05 vs. non-treated group, #P , 0.05 vs. evodiamine-treated group.

Article Snippet: Rabbit antibodies for phospho-eNOS at Ser1179, phosphoCaMKII at Thr286, phospho-serine/threonine and mouse antibody for phospho-Akt at Ser473 were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Incubation, Transfection, Western Blot, Griess Assay

Journal: Cardiovascular research

Article Title: Molecular mechanisms of activation of endothelial nitric oxide synthase mediated by transient receptor potential vanilloid type 1.

doi: 10.1093/cvr/cvr104

Figure Lengend Snippet: Figure 5 Deletion of TRPV1 impairs TRPV1 ligand-induced eNOS activation. (A) Eight-week-old male WT mice were killed after intraperitoneal (ip) injection with evodiamine (3 mg/kg) for the indicated times. The control mice received the same amount of DMSO/alcohol. (B) WT mice were pre- treated with CGRP antagonist, SB268262 (10 mg/kg) for 1 h, followed by evodiamine administration for 4 h. (C) WT and TRPV12/2 mice were killed after evodiamine injection (3 mg/kg) for 4 h. (D) Mouse aortic ECs (MAECs) were isolated from WT and TRPV12/2 mice and treated with evodiamine (1 mmol/L) for 15 min. Tissue or cellular extracts from aortas were immunoblotted with antibodies to phosphorylated eNOS at Ser1179, eNOS protein, phosphorylated Akt at Ser473, Akt protein, phosphorylated CaMKII at Thr286, and CaMKII protein. Data are mean+ SD from five mice. *P , 0.05 vs. WT mice without evodiamine treatment, #P , 0.05 vs. WT mice with evodiamine treatment.

Article Snippet: Rabbit antibodies for phospho-eNOS at Ser1179, phosphoCaMKII at Thr286, phospho-serine/threonine and mouse antibody for phospho-Akt at Ser473 were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Injection, Control, Isolation

Journal: International journal of cancer

Article Title: MAP kinase pathway gene copy alterations in NRAS/BRAF wild-type advanced melanoma.

doi: 10.1002/ijc.29970

Figure Lengend Snippet: Figure 1. NRAS/BRAF/cKIT wild-type melanoma metastases can show activation of the MAP kinase (MAPK) signaling pathway. (a) BRAFV600E, MEK1/2, MEK2, phosphorylated MEK (p-MEK), ERK1/2, ERK2 and phosphorylated ERK (p-ERK) expression levels in a representative NRASwild-type/ BRAFwild-type/cKitwild-type metastatic melanoma specimen. 1003 and 4003 magnifications are shown for each of the immunohistochemical (IHC) stainings. (b) IHC analyses of members of the MAPK signaling pathway in NRAS mutant (NRAS), BRAFV600E mutant (BRAF) and NRASwild-type/ BRAFwild-type/cKitwild-type (WT) melanoma subgroups. Y-axis shows the overall IHC score (0–12) calculated based on the quantity and intensity of the staining. Asterisks show p-values <0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: 4 mm sections of formalin-fixed paraffin-embedded melanoma metastases were stained with antibodies against BRAFV600E (Spring Bioscience, Cat. No. E19290), total MEK (MEK1/2, Cell Signaling, Cat. No. 4694), MEK2 (Abcamab32517), phosphorylated MEK (Phospho-MEK1/2 [Ser221], Cell Signaling, Cat. No. 2338), total ERK (p44/42 MAPK [Erk1/2], Cell Signaling, Cat. No. 4695), ERK2 (Abcamab32081) and phosphorylated ERK (Phospho-p44/42 MAPK [Erk1/2][Thr202/Tyr204], Cell Signaling, Cat. No. 4376) using standard protocols.

Techniques: Activation Assay, Expressing, Immunohistochemical staining, Mutagenesis, Staining