Journal: Immunity

Article Title: Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses

doi: 10.1016/j.immuni.2020.11.015

Figure Lengend Snippet:

Article Snippet: Cynomolgus CD32b/FCGR2B/Fc gamma RIIB Gene ORF cDNA clone expression plasmid , Sino Biological , Cat#CG90014-ACG.

Techniques: Purification, Recombinant, Enzyme-linked Immunosorbent Assay, RNA Detection, Enzyme-linked Immunospot, Bicinchoninic Acid Protein Assay, Luciferase, Transgenic Assay, Plasmid Preparation, Expressing, Software, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Cyclin-dependent Kinase 5 (Cdk5) at Tyr-15 Is Inhibited by Cdk5 Activators and Does Not Contribute to the Activation of Cdk5

doi: 10.1074/jbc.m113.501148

Figure Lengend Snippet: FIGURE 6. Tyr-15 phosphorylation of Cdk5 in neurons treated with Sema3A, Ephrin-A1, or BDNF. A, immunoblots of neuronal lysates with anti- phospho-Tyr-15 Cdk5 (pY15). Mouse cortical neurons prepared from wild- type (/) or Cdk5 knockout (/) mouse brains were immunoblotted with antibodies to Cdk5 C8, anti-phospho-Tyr-15 of Cdk5 (sc) and (ab), and actin. B, an immunoblot of anti-Cdk5 C8 immunoprecipitates with anti-phospho- Tyr-15 (sc). Cdk5 was immunoprecipitated (IP) from the extract (Ext) of mouse brains at embryonic day18.5, which was treated with Na3VO4 (V) and ATP. The supernatant (sup) or immunoprecipitate (ppt) was immunoblotted with anti- Cdk5 C8 or control IgG, followed by immunoblotting with anti-phospho- Tyr-15 Cdk5 (sc) (top panel) or anti-Cdk5 C8 (bottom panel). C, phospho-Cdk5 at Tyr-15 did not coimmunoprecipitate with anti-p35. Cultured neuronal lysates were immunoprecipitated with anti-Cdk5 C8 or anti-p35 C19, and Cdk5 phosphorylated at Tyr-15 was detected with anti-phospho-Tyr-15 Cdk5 (sc). D, Tyr-15 phosphorylation in neurons treated with Sema3A, Ephrin-A1, or BDNF.MousecorticalneuronsweretreatedwithSema3A,Ephrin-A1(Eph-A1), BDNF, or 100 M pervanadate (PV) for the indicated times. Cell lysates were immunoblotted with antibodies to Cdk5 (third panel), p35 (fourth panel), anti- phospho-SFK (fifth panel), or phospho-Thr-202/Tyr-204 of ERK (p-MAPK, sixth panel). Phosphorylation of Tyr-15 on Cdk5 was detected with anti-phospho- Tyr-15 Cdk5 (sc) (first panel) after immunoprecipitation with anti-Cdk5 C8 (second panel). Lane 10 is a molecular weight (MW) marker. The asterisk indi- cates carbonic anhydrase at 32.2 kDa. Lanes 11 and 12 are Cdk5 coexpressed with or without caFyn in HEK293 cells for reference. E, the effect of Sema3A on neurite retraction. Neurites of mouse cortical neurons expressing EGFP were observed by time-lapse imaging at intervals of 5 min after addition of Sema3A. Scale bar 5 m. F, kinase activity of Cdk5 after Sema3A treatment. Cdk5 was prepared from cultured neurons treated with Sema3A for 30 min by immunoprecipitation, and its kinase activity was measured by phosphoryla- tion of histone H1. Data are mean S.E. (n 3). ns, not significant; Student’s t test. Ctrl, control. G, the protein ratio of p35 and Cdk5 in COS-7 cells and mouse brain extract. FLAG-Cdk5 and FLAG-p35 were transfected into COS-7 cells using 1 g of plasmid for each of Cdk5 and p35, the same as in Fig. 2. Immunoblotting was performed by adjusting Cdk5 approximately (bottom panel). FLAG-p35 expressed in COS-7 cells is indicated by an arrowhead, and p35 in the brain extract (BrE) is indicated by an arrow (top panel). H, the effect of p35 at the low expression levels (similar to the brain extract) on Tyr-15 phosphorylation of Cdk5. COS-7 cells were transfected with FLAG-Cdk5 and caFyn with or without FLAG-p35 as shown in Fig. 2A, except for the reduced plasmid amount of p35 (1/40). Phosphorylation of Tyr-15 in Cdk5 was detected by immunoblotting with antibodies to phospho-Tyr-15 of Cdk5 (ab). Blotting of Cdk5, p35, Fyn, and actin is also shown.

Article Snippet: Semaphorin 3A (Sema3A) and Ephrin-A1 were from R&D Systems (Pune, Maharashtra, India).

Techniques: Phospho-proteomics, Western Blot, Knock-Out, Immunoprecipitation, Control, Cell Culture, Molecular Weight, Marker, Expressing, Imaging, Activity Assay, Transfection, Plasmid Preparation

Journal: NPJ Precision Oncology

Article Title: Comprehensive RNA analysis of CSF reveals a role for CEACAM6 in lung cancer leptomeningeal metastases

doi: 10.1038/s41698-021-00228-6

Figure Lengend Snippet: A Robust rPCA representation of all single cells included in this study ( n = 197) from 4 patients. Cells are colored by sample ID. B – D Expression of characteristic cell-type-specific genes overlaid on the robust rPCA plot ( P < 2 × 10 −16 , t -test). E Cell-free CEACAM6 RNA level in CSF validated by qPCR. Results are shown as the mean ± standard deviation (SD) performed with three technical replicates.

Article Snippet: In vitro knockdown of the CEACAM6 gene was achieved by the treatment of A549 cells with 250 nM of CEACAM6 -specific small interfering RNA (siRNA) oligos, and non-targeting control siRNA (ON-TARGETplus SMARTpool and ON-TARGETplus Non-targeting Pool, accordingly, Dharmacon, Lafayette, CO, USA) for 72 h. To achieve overexpression of CEACAM6 protein in H460 cells, cells were transfected with full-length ready-to-use CEACAM6 cDNA (Sino Biological Inc, Beijing, China) using Lipofectamine™2000 (Invitrogen™) according to the manufacturer’s protocol.

Techniques: Expressing, Standard Deviation

Journal: NPJ Precision Oncology

Article Title: Comprehensive RNA analysis of CSF reveals a role for CEACAM6 in lung cancer leptomeningeal metastases

doi: 10.1038/s41698-021-00228-6

Figure Lengend Snippet: A A549 lung adenocarcinoma cells natively expressed high levels of CEACAM6 as opposed to H460 cells that showed undetectable levels of CEACAM6 , as measured by qPCR. A549 cells were treated for 72 h with siCEACAM6 failed to migrate toward FBS. B Western blot analysis confirmed CEACAM6 knockdown in A549 cells following treatment with siRNA. C qPCR analysis confirmed CEACAM6 knockdown in A549 cells following 72 h knockdown with siRNA. D Quantitative analysis and representative images of decreased migration of A549 cells following knockdown of CEACAM6 , as compared with siCtrl-treated cells (normalized to untreated cells). E Western blot and F qPCR confirmed elevated CEACAM6 levels in H460 cells following plasmid transfection. G Quantitative analysis and representative images showed increased cell migration following overexpression of CEACAM6, as compared with H460 cells. Student’s t -test was used to evaluate the statistical significance of the difference between groups in D and G . Scale bar = 400 µm in D and G images. Results are shown as the mean ± standard deviation (SD) of three independent assays.

Article Snippet: In vitro knockdown of the CEACAM6 gene was achieved by the treatment of A549 cells with 250 nM of CEACAM6 -specific small interfering RNA (siRNA) oligos, and non-targeting control siRNA (ON-TARGETplus SMARTpool and ON-TARGETplus Non-targeting Pool, accordingly, Dharmacon, Lafayette, CO, USA) for 72 h. To achieve overexpression of CEACAM6 protein in H460 cells, cells were transfected with full-length ready-to-use CEACAM6 cDNA (Sino Biological Inc, Beijing, China) using Lipofectamine™2000 (Invitrogen™) according to the manufacturer’s protocol.

Techniques: Western Blot, Migration, Plasmid Preparation, Transfection, Over Expression, Standard Deviation