anti human scf antibody  (R&D Systems)

Journal: Molecular Therapy Oncology

Article Title: Development of multivalent CAR T cells as dual immunotherapy and conditioning agents

doi: 10.1016/j.omton.2025.200944

Figure Lengend Snippet: Overview of ELECTRIC CAR (A) Schematic of ELECTRIC CARs. SCF is N terminus, followed by a (G 4 S) 3 linker, TPO, a second (G 4 S) 3 linker, FLT3LG, a CD28 hinge, transmembrane, and intracellular (IC) signaling domain, and CD3ζ. (B) ColabFold-predicted structure of extracellular domain of ELECTRIC CAR, with representative binding modes to the KIT, MPL, and FLT3. (C) Comparison of in vitro T cell expansion after viral transduction to Mock (non-transduced) T cells (two-way ANOVA; ∗∗∗∗ p < 0.0001). (D) Transduction efficiency (left) and ELECTRIC CAR MFI (right) of transduced CAR T cells 8 days after viral transduction. (E) Representative flow cytometry plot (left) of anti-hSCF detection by murine anti-IGG1-PE fluorescent antibody (right). (F–H) Nalm-6 (N6) B-ALL cells were transduced with a virus encoding the extracellular and transmembrane domains of KIT (F), MPL (G), and FLT3 (H). (I–K) Monovalent natural ligand CARs expressing SCF, TPO, or FLT3LG and ELECTRIC CARs were co-cultured with GFP+ N6 cells overexpressing (OE) KIT (I), MPL (J), or FLT3 (K) and N6 GFP expression was measured over time via Incucyte Live Imaging. (L–N) ELECTRIC CAR T cells were co-cultured across a range of E:T ratios with N6 KIT OE (L), N6 MPL OE (M), and N6 FLT3 OE (N) and monitored via Incucyte Live Imaging. (O) Monovalent natural ligand CARs expressing SCF, TPO, or FLT3LG and ELECTRIC CARs were co-cultured with a heterogeneous mixture of N6 KIT OE, N6 MPL OE, and N6 FLT3 OE (1:1:1 ratio of KIT:MPL:FLT3) and monitored via Incucyte Live Imaging. (P) Monovalent natural ligand CARs expressing SCF, TPO, or FLT3LG and ELECTRIC CARs were co-cultured with N6 KIT OE, N6 MPL OE, and N6 FLT3 OE at 1:1 E:T ratios for 24 h, and supernatants were harvested for cytokine production measurement by ELISA. Cytokine production was compared between Mock and CAR T cells for each type of target cell (two-way ANOVA, Tukey’s multiple comparison test). For I–P, data are mean ± s.e.m. of triplicate wells.

Article Snippet: Triplekine CARs were detected with an anti-human SCF antibody (Catalog #MAB655, R&D Systems, Minneapolis, MN) followed by an anti-IGG1 PE antibody (Clone: RMG1-1, BioLegend, San Diego, CA) or an anti-G 4 S Linker antibody (Clone: E702V, Cell Signaling Technology, Danvers, MA).

Techniques: Binding Assay, Comparison, In Vitro, Transduction, Flow Cytometry, Virus, Expressing, Cell Culture, Imaging, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: Endothelial HIF α /PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension

doi: 10.1172/jci.insight.162449

Figure Lengend Snippet: Immunohistochemical staining for ( A ) Beclin1, ( B ) LC3B, and ( C ) p62 as markers of autophagy in combination with SMA, Cx40, and DAPI in lungs of human control and IPAH patients ( n = 4). Closeup of boxed region in A is shown to the right. Scale bars: 50 μm.

Article Snippet: Primary antibodies used for Western blot analysis were rabbit anti–HIF1-α (1:500; Novus, NB100-449), rabbit anti–HIF2-α (1:500; Novus, NB100-122), rabbit anti-Beclin1 (1:500; Novus, NB110-87318), rabbit anti-LC3B (1:500; Cell Signaling Technology, 2775), or rabbit anti-p62 (1:500; Abcam, ab109012).

Techniques: Immunohistochemical staining, Staining

Journal: JCI Insight

Article Title: Endothelial HIF α /PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension

doi: 10.1172/jci.insight.162449

Figure Lengend Snippet: ( A ) qRT-PCR analysis of autophagy-related gene products and HIF1A , HIF2A , and PDGFB from lung lysates of patients with IPAH compared with that of control individuals ( n = 6). ( B ) Western blots of lung lysates of IPAH patients and controls probed for Beclin1, LC3B, p62, and GAPDH. ( C ) Densitometry of protein bands shown in B relative to GAPDH and normalized to control ( n = 4). ( D ) Acta2-CreER T2 ROSA26R Zs/+ mice were induced with tamoxifen, rested for 5 days, exposed to normoxia or hypoxia for 21 days or to hypoxia for 21 days, followed by re-normoxia for 10 days. Lung Zs + cells were isolated by FACS, and the expression of autophagy genes Atg5 , Atg7 , Becn1 , and Map1lc3b was analyzed by qRT-PCR and normalized to normoxia. n = 3 mice (1 male, 2 females) per experimental group. Significance assessed by 2-tailed Student’s t test ( A and C ) or multifactor ANOVA with Tukey’s multiple-comparison test ( D ).

Article Snippet: Primary antibodies used for Western blot analysis were rabbit anti–HIF1-α (1:500; Novus, NB100-449), rabbit anti–HIF2-α (1:500; Novus, NB100-122), rabbit anti-Beclin1 (1:500; Novus, NB110-87318), rabbit anti-LC3B (1:500; Cell Signaling Technology, 2775), or rabbit anti-p62 (1:500; Abcam, ab109012).

Techniques: Quantitative RT-PCR, Western Blot, Isolation, Expressing, Comparison

Journal: JCI Insight

Article Title: Endothelial HIF α /PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension

doi: 10.1172/jci.insight.162449

Figure Lengend Snippet: ( A ) Experimental strategy for B . ( B ) Human PAECs were exposed to normoxia or hypoxia (3% O 2 ) for 16 hours, and the conditioned medium was collected and pretreated with either anti–PDGF-B blocking antibody or IgG isotype control for 1 hour. Human PASMCs were incubated with the pretreated PAEC-conditioned medium in normoxic conditions for 48 hours, and then qRT-PCR was used to assess mRNA levels of ATG5 , ATG7 , BECN1 , and MAP1LC3B in the PASMCs. Transcript levels relative to 18S rRNA were normalized to normoxia PAEC medium treated with IgG (dashed line). n = 3. Significance assessed by multifactor ANOVA with Tukey’s multiple-comparison test. ( C ) Experimental strategy for D and E . ( D ) Cdh5-CreER T2 Pdgfb fl/fl mice were exposed to hypoxia for 35 days and tamoxifen (1 mg/day) was or was not administered on hypoxia days 17–21. Vibratome lung sections were stained for Beclin1, SMA, and nuclei (DAPI). Closeups of boxed region are shown below. Arrowheads indicate Beclin1 + SMA + cells. ( E ) Quantification of the percentage of distal arteriole SMCs that are Beclin1 + . n = 3 mice (2 males, 1 female) per experimental group, 3–4 arterioles analyzed per mouse. Significance assessed by 2-tailed Student’s t test. Scale bar: 20 μm.

Article Snippet: Primary antibodies used for Western blot analysis were rabbit anti–HIF1-α (1:500; Novus, NB100-449), rabbit anti–HIF2-α (1:500; Novus, NB100-122), rabbit anti-Beclin1 (1:500; Novus, NB110-87318), rabbit anti-LC3B (1:500; Cell Signaling Technology, 2775), or rabbit anti-p62 (1:500; Abcam, ab109012).

Techniques: Blocking Assay, Incubation, Quantitative RT-PCR, Comparison, Staining

Journal: JCI Insight

Article Title: Endothelial HIF α /PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension

doi: 10.1172/jci.insight.162449

Figure Lengend Snippet: ( A ) Experimental strategy for B – D . ( B ) Acta2-CreER T2 ROSA26R Zs/+ mice were exposed to hypoxia or normoxia for 31 days and STI571 was administered at 0, 50, or 100 mg/kg/d by daily intraperitoneal injections on hypoxia days 21–31. Lung Zs + SMCs were isolated by FACS, and expression levels of Atg5 , Atg7 , Becn1 , and Map1lc3b with hypoxia relative to normoxia, no STI571 were analyzed by qRT-PCR. n = 3 mice (2 males, 1 female) per experimental group. ( C ) Vibratome lung sections were stained for Beclin1, SMA, and nuclei (DAPI). Closeups of boxed regions are shown below. ( D ) Quantification of the percentage of distal arteriole SMCs that are Belclin1 + . n = 3 mice (1 male, 2 female) per experimental group, 3–4 arterioles analyzed per mouse. Significance assessed by multifactor ANOVA with Tukey’s multiple-comparison test ( B and D ). Scale bar: 20 μm.

Article Snippet: Primary antibodies used for Western blot analysis were rabbit anti–HIF1-α (1:500; Novus, NB100-449), rabbit anti–HIF2-α (1:500; Novus, NB100-122), rabbit anti-Beclin1 (1:500; Novus, NB110-87318), rabbit anti-LC3B (1:500; Cell Signaling Technology, 2775), or rabbit anti-p62 (1:500; Abcam, ab109012).

Techniques: Isolation, Expressing, Quantitative RT-PCR, Staining, Comparison