Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Time- and dose-dependent inhibition of NAC on the intracellular domain of Notch3 (N3IC), but not Notch1 (N1IC). HeLa cells were treated with NAC (2-10 mM) for 0-24 h. B. Dose-dependent inhibition by NAC (0-10 mM, 6 h) on the protein expression of N3IC in HeLa cells. C. NAC treatment (5 mM, 0-12 h) reduces protein levels of N3IC and extracellular domain of Notch 3 (N3EC) but not full length Notch 3 precursor (N3FL) in HeLa cells. Densitometry quantifications of the protein bands were shown after normalization with their respective β-actin levels. Data are presented as means ± SE, n=3. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Inhibition, Expressing

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (2-10 mM, 0-24 h) decreases Hes1 and HRT1 protein levels in HeLa cells. B. NAC treatment (0-10 mM for 6 h or 5 mM for 0-12 h) decreases Hes1 and HRT1 mRNA expression in HeLa cells. The mRNA expression of NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. C. NAC treatment (0-10 mM, 12 h) inhibits Hes1 reporter activity in HeLa cells. The luciferase activity in NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. D. Notch3 siRNA knockdown reduces Hes1 and HRT1 levels in HeLa cells. Protein levels were determined 2 days after siRNA transfection. siCtrl, scramble siRNA; siNotch3, Notch3 siRNA. Protein densitometry quantifications were shown after normalization with β-actin levels. Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Activity Assay, Luciferase, Knockdown, Transfection

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Pre-treatment with a γ-secretase inhibitor, DAPT (20 μM, 30 min), had no effect on NAC-induced (5 mM, 0-12 h) decrease in N3IC protein expression in HeLa cells. B. NAC treatment (5 mM, 0-12 h) did not affect Notch3 mRNA expression in HeLa cells. C. Pre-treatment with NH 4 Cl (25 mM, 1 h), but not lactacystin (10 μM, 30 min), reversed NAC-induced (5 mM, 0-12 h) decrease of N3IC protein levels in HeLa cells. D. NAC treatment did not affect levels of exogenously expressed Notch3 active intracellular domain (N3ICD). HeLa cells were transfected with vectors expressing N3ICD or N3FL for 24 h, followed by treatment with NAC (5 mM, 0-12 h). E. Subcellular analysis of Notch3 protein levels following NAC treatment (5 mM, 6 h) in HeLa cells. Protein levels of N3FL, N3EC and N3IC in cytosolic, nuclear and membrane fractions were determined. Successful fractionation was evidenced by using the marker proteins GAPDH, cyclin B1, and Na + , K + -ATPase. N3FL, N3EC and N3IC denoted Notch3 full length, extracellular domain and intracellular domain, respectively. Protein densitometry quantifications were shown after normalization with β-actin (A, C, D) or their respective cellular compartment markers (E). Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Transfection, Membrane, Fractionation, Marker

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: N3ICD overexpression rescues NAC-induced inhibition of proliferation (A), migration (B), and invasion (C). A. Numbers of EV- and N3ICD-transfected cells were counted at 12-48 h after NAC treatment (0-10 mM, left panel). *, p < 0.05 compared with the EV-transfected cells within the same treatment and time point. B. Results of the wound healing assay (left panels) were expressed as the migration index (the distance migrated relative to the initial scraped gap) and that of EV-transfected cells without NAC treatment was set as 100% (middle panel). C. Cells per field on the insert membrane were imaged (left panels) and counted (middle panel). B and C: *, p < 0.05 compared with no NAC treatment; #, p < 0.05 compared with the EV-transfected cells within the same treatment. Percent rescue (A-C, right panels) after N3ICD expression was calculated by dividing the net change after NAC treatment in N3ICD-transfected cells by that in EV-transfected cells. Notch3 siRNA knockdown inhibits cell proliferation D. , migration E. , and invasion F. as assessed by the same approaches described above. Representative images for migration and invasion were shown. *, p < 0.05 compared with the siCtrl-transfected cells. All data are presented as mean ±SE, n=3. I, the initial seeded cell number. EV, empty vector; N3ICD, Notch3 active intracellular domain; siCtrl, scrambled siRNA; siNotch3, Notch3 siRNA.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Over Expression, Inhibition, Migration, Transfection, Wound Healing Assay, Membrane, Expressing, Knockdown, Plasmid Preparation

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (5 and 10 mM, 0-24 h) decreases N3IC protein levels in HCC1937 cells. Expression of exogenous N3ICD rescues NAC-induced inhibition of proliferation B. , migration C. , and invasion D. , and Notch3 siRNA knockdown inhibits proliferation E. , migration F. , and invasion G. in HCC1937 cells. Quantifications, sample size, statistics, and abbreviations for protein levels, proliferation, migration, and invasion assays were as described in Figure & legends.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Inhibition, Migration, Knockdown

Journal: Cancer Research

Article Title: Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

doi: 10.1158/0008-5472.can-10-0690

Figure Lengend Snippet: Figure 1. Retinal lesions and ASCs are caused by Notch3 signaling. A, virus is microinjected into the ventricles of an E10.5 mouse embryo. At the time of injection, the forebrain and optic cup are a continuous structure, allowing viral infection of the anlage giving rise to the retina and optic nerve. Viral particles are released into the amniotic sac near the injection site on needle withdrawal and infect the developing lens. B, E17.5 retina in a N3CLE-injected animal with a PLAP-positive clone (i). H&E staining showed that N3CLE-induced retinal lesions can span 1 (ii) or all 3 layers (iii) of the retina. Retinal lesions contained BIII- tubulin–positive (iv) and GS-positive (v) cells that were often double positive for nuclear Notch3 (N3). C, Pax6/Chx10 double-positive cells in retinal lesions (arrowheads). D, i, white cataractous lenses developed in N3CLE-injected animals. ii, the ALE (arrowhead) expanded into multiple epithelial layers (arrow) and contained mitotic bodies in the N3CLE-induced ASCs (inset). iii, areas of hyperproliferation were PLAP positive and contained Ki67-positive cells (DAPI counterstain). Original magnifications: 20 (Bi, Biv, and C), 40 (Bv and Dii), 200 (Bii, Biii, and Di).

Article Snippet: Primary antibodies used were a-Notch3 (rabbit, 1:1,000; Santa Cruz Biotechnology), a-Pax6 (mouse, 1:400, Millipore Corporation), a-Chx10 (sheep, 1:100, Exalpha Biologicals), a-GFAP (rabbit, 1:1,000; DAKO), a-Nestin (chicken, 1:250; Aves Labs, Inc.), and Hes5 (rabbit, Gaiano Laboratory, only recognizes overexpression).

Techniques: Virus, Injection, Infection, Staining

Journal: Cancer Research

Article Title: Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

doi: 10.1158/0008-5472.can-10-0690

Figure Lengend Snippet: Figure 2. Invasive choroidal tumors and retinal glial lesions arise following Notch3 activation. N3CLE-induced cellular choroidal tumors in the adult eye were PLAP positive (A), pigmented, and arose in the choroidal layer beneath an intact retinal pigment epithelium (Bi, arrow). Many choroidal tumors invaded outward through breaks in the sclera (Bi, arrowheads) and could diffusely infiltrate periocular tissues. Choroidal tumors contained nuclear N3 and Ki67-positive cells (Bii and Biii). C and D, N3CLE-injected animals contained PLAP-positive retinal glial lesions (C), GFAP-positive (Dii), and GS-positive (Diii). Original magnifications: 10 (A), 20 (Bi), 40 (Bii and Diii), 63 (Biii, Di, and Dii), 64 (Bii), 200 (C).

Article Snippet: Primary antibodies used were a-Notch3 (rabbit, 1:1,000; Santa Cruz Biotechnology), a-Pax6 (mouse, 1:400, Millipore Corporation), a-Chx10 (sheep, 1:100, Exalpha Biologicals), a-GFAP (rabbit, 1:1,000; DAKO), a-Nestin (chicken, 1:250; Aves Labs, Inc.), and Hes5 (rabbit, Gaiano Laboratory, only recognizes overexpression).

Techniques: Activation Assay, Injection

Journal: Cancer Research

Article Title: Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

doi: 10.1158/0008-5472.can-10-0690

Figure Lengend Snippet: Figure 3. N3CLE induced formation of glial tumors along the optic nerve. A, glial tumors (arrowheads) formed along the optic nerve (arrow) in N3CLE-injected animals and contained mitotic bodies (ii, inset). B, the optic nerve tumors expressed GFAP and Notch3 and invaded orbital tissues surrounding the optic nerve. Some glial tumors involved both the retina (arrowhead) and optic nerve (arrow). C, i, glial tumors contained Ki67-positive proliferating cells. Nestin-positive and Nestin/GFAP-positive cells within the lesions coexpressing Hes5 or Notch3, respectively (Cii and D). Original magnifications: 63 (C and D), 100 (Ai and Bi), 200 (Ai, Aii, and Bii), 400 (Aii, inset).

Article Snippet: Primary antibodies used were a-Notch3 (rabbit, 1:1,000; Santa Cruz Biotechnology), a-Pax6 (mouse, 1:400, Millipore Corporation), a-Chx10 (sheep, 1:100, Exalpha Biologicals), a-GFAP (rabbit, 1:1,000; DAKO), a-Nestin (chicken, 1:250; Aves Labs, Inc.), and Hes5 (rabbit, Gaiano Laboratory, only recognizes overexpression).

Techniques: Injection

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Schematic representation of Notch signaling. (1) Furin (S1 cleavage) cleaves the NOTCH3 precursor protein in the Golgi system, resulting in a non-covalently bound heterodimeric protein that is transported to the cell surface. (2) A mechanical traction force is applied to the NOTCH3 ECD when a Notch ligand binds to the EGF repeats 10-11, exposing the extracellular NRR near the cell membrane, which consists of LNR and the heterodimerization domain (in green). Subsequently, ADAM17 cleaves the C-terminal portion of the heterodimerization domain (S2-cleavage). (3) The NEXT, which is made up of a RAM domain, many ANK domains, a PEST domain, and a transmembrane domain, is cleaved by the γ-secretase (S3-cleavage) releasing the N3ICD. (4) The N3ICD binds to the CSL complex protein and together with the co-activator Mastermind-like (MAM) trigger downstream gene transcription in the nucleus. (5) The NOTCH3 ECD and ligand are normally endocytosed by the ligand expressing cell and is degraded in the lysosome. B) Schematic representation of NOTCH3 cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) mutations. NOTCH3 ECD contains 34 EGF repeat domains, each of which has 6 cysteine residues (WT). Mutations in CADASIL change the number of cysteines to an uneven number of cysteines (Mutant). These unpaired cysteines residues result in incorrect EGF repeat folding, irregular protein folding which leads to an enhanced NOTCH3 ECD multimerization. Distribution of the cysteine-altering mutations that cause CADASIL are shown. In the CADASIL mutant NOTCH3 ECD, the endocytosis is hampered, and NOTCH ECD remains outside of the VSMC and starts to accumulate and aggregate around the vessels. ADAM17, a disintegrin and metalloproteinase domain-containing protein 17; ANK, ankyrin repeats; EGF, epidermal growth factor; HD, heterodimerization domain; LNR, Lin-Notch repeats; PEST, proline (P), glutamic acid (E), serine (S) and threonine (T) degradation domain; RAM, Rbp-associated molecule domain; TM, transmembrane domain.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Membrane, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Schematic representation of NOTCH3 and NOTCH3 EGF 1-5 . NOTCH3 represents the full-length protein, and NOTCH3 EGF 1-5 represents the NOTCH3 protein with exon 1 to 5 fused with a myc-His-Tag at the C-terminus used for purification of the aggregated protein. B) Western blot of the NOTCH3 EGF 1-5 WT and R133C purified protein. The eluate fractions were visualized by western blot using an α-myc antibody. C) Western blot of NOTCH3 EGF 1-5 WT and R133C aggregated proteins. The incubated fractions of NOTCH3 EGF 1-5 WT and R133C were visualized on a western blot using an α-myc antibody. The purified proteins and the aggregates were verified after 1-5 days incubation by western blot using α-myc ab under non-reducing conditions.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Purification, Western Blot, Incubation

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Schematic and work plan of the subcutaneous active immunization on the TgN3R182C 150 mouse model. B) Antibody titre validation of serum from TgN3R182C 150 CADASIL mice immunized with NOTCH3 EGF 1-5 aggregates (vaccinated) and PBS (sham) at 4, 5 and 7 months old. A direct ELISA with NOTCH3 aggregate-coated plates and different dilutions of serum was performed.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Biomarker Discovery, Direct ELISA

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Representative images of TgN3R182C 150 , sham- and NOTCH3 EGF 1-5 - immunized mice at 7 months of age and TgN3R182C 150 at 18 months of age. Representative images show brain arteries of TgN3R182C 150 (7 and 18 months), sham and NOTCH3 EGF 1-5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an α-SMA antibody (green). B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF 1-5 - immunized, sham and non-vaccinated TgN3R182C 150 mice at 7 months of age. NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per vessel increases significantly in the TgN3R182C 150 mice at 18 months of age versus NOTCH3 EGF 1-5 - immunized, sham and non-vaccinated TgN3R182C 150 mice at 7 months of age. (*p < 0.05, **p < 0.01, ns= non-significant). Scale bar =20µm.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Staining

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: Quantitative real-time PCR analysis of the Notch downstream target genes NOTCH3, Hes1, Hey1 and Nrip2 on TgN3R182C150 mice at 5 and 12 months of age.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Representative images of TgN3R182C 150 , sham- and NOTCH3 EGF 1-5 - immunized mice at 3, 7 and 18 months of age. Representative images show brain arteries and capillaries of TgN3R182C 150 , sham and NOTCH3 EGF 1-5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an anti-perlecan antibody (green). B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3-ECD stained area and average size per vessel revealed a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non-vaccinated 3 months old TgN3R182C 150 (n=3) and 7 months old TgN3R182C 150 (n=6) mice and 18 months old TgN3R182C 150 (n=3). Quantification of NOTCH3-ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3-ECD stained area and average size per vessel revealed a significant decrease in NOTCH3-ECD deposition in brain arteries and capillaries between NOTCH3 EGF 1-5 - immunized (n=11), sham (n=9) and non-vaccinated TgN3R182C 150 (n=6) mice. (*p < 0.05, **p < 0.01, ****p < 0.0001, ns= non-significant). Scale bar =20µm.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Staining

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: Quantification of human NOTCH3 ECD protein present in the whole blood serum of sham, immunized and non-vaccinated TgN3R182C 150 mice (at 3 and 7 months old). A) NOTCH3 ECD was detected in the whole blood serum of the non-treated TgN3R182C 150 mice at three months of age and further increased at seven months of age. B) NOTCH3 ECD in the TgN3R182C 150 mice was significantly reduced in the vaccinated TgN3R182C 150 mice. Statistical analysis was performed using unpaired t test with Welch’s correction. P < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques:

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Immunostaining for smooth muscle actin (ASMA) revealed that there were no significant differences in the composition of the smooth muscle cell coating of vessels in the retinal vasculature in WT (C57Bl6/J) versus TgN3R182C 150 mice at 7 months of age. B) Immunostaining for smooth muscle actin (ASMA) shows no significant differences in the composition of the smooth muscle cell coating of vessels in the retinal vasculature in NOTCH3 EGF 1-5 - vaccinated versus sham-vaccinated TgN3R182C 150 mice. C) Immunostaining for smooth muscle actin (ASMA) shows an extensive loss of VSMC in the Notch3 -/- mice when compared to a WT (C57Bl6/J) at 3 months of age. Scale bar =50µm.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Immunostaining

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Representative images of TgN3R182C 150 , sham- and NOTCH3 EGF1-5 - immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an antibody against microglia (Iba1, green). B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF1-5 - immunized (n=11), sham (n=9) and non-vaccinated TgN3R182C 150 (n=6) mice at 7 months of age. (ns= non-significant). Scale bar =20µm.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Staining

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: NIH3T3 cells were transfected with the control, wild type NOTCH3, or NOTCH3 R182C plasmids, as well as the β-gal and 12XCSL-luc reporter plasmids and cultured on immobilized jagged2 (Jag2) in the presence of DMSO or DAPT (n=3 and two technical replicates). Statistical analysis was performed using 2-way ANOVA followed by Tukey’s multiple comparisons tests. P < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, ns= non-significant). RLU, relative luminescence units.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Transfection, Control, Cell Culture