Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Flicr , a long noncoding RNA, modulates Foxp3 expression and autoimmunity

doi: 10.1073/pnas.1700946114

Figure Lengend Snippet: Flicr is expressed specifically in Tregs. Flicr expression in various contexts. (A and B) ImmGen mouse immunocyte (39) or GNF mouse organ microarray compendia (40) (dashed lines: background level). (C) During thymic Treg differentiation. (Left) ImmGen data. (Right) RNA-Seq analysis of CD4+ SP thymocytes sorted as shown; y-axis, transcripts per million (tpm). (D) Tissue Tregs. Each point is an individual mouse. Sp, spleen; Col, colonic lamina propria; Panc, pancreas; Adip. tissue, visceral adipose tissue; Muscle, injured muscle at 4 d postcardiotoxin injection. (E) After activation in vitro with anti-CD3/CD28 beads (42) (Left) or in vivo (41) (Right). Rest., resting CD44loCD62Lhi; Act., activated CD44hiCD62Llo Tregs; rpkm, reads per kb per million reads. (F) In vitro converted induced Tregs (TCR activation with IL-2 and TGF-β) (43). (G) Ex vivo Helios−RORγ+ colonic peripheral Tregs (44). (H) In Tconvs transduced with Foxp3 or control retrovectors (11). (I) In Treg-like cells of mice with Foxp3 inactivation by Gfp insertion (Foxp3ΔGfp) (45).

Article Snippet: Total CD4 T cells from spleen and subcutaneous lymph nodes were isolated by magnetic negative selection (Dynabeads Untouched Mouse CD4 T-Cell Kit, 11415D; Thermo Fisher Scientific) and activated in vitro in 200 µL of cRMPI for 72 h with anti-CD3/CD28 beads in a 96-well plate (0.1 M cell/well, 1:1 bead:cell ratio; Dynabeads Mouse T-Activator CD3/CD28; Thermo Fisher Scientific) and 50 U/mL IL-2 (recombinant human IL-2, 212–12; Peprotech).

Techniques: Expressing, Microarray, RNA Sequencing Assay, Injection, Activation Assay, In Vitro, In Vivo, Ex Vivo, Transduction

Journal: BMC Research Notes

Article Title: Development of repeatable arrays of proteins using immobilized DNA microplate (RAPID-M) technology

doi: 10.1186/s13104-015-1637-3

Figure Lengend Snippet: A schematic representation of the RAPID-M technology. The modified T7Term primer was mixed with the immobilization beads in a PCR microwell plate ( a ) to create beads-immobilized T7Term primer ( b ). The target DNA is directly immobilized to the beads by PCR amplification using beads-immobilized T7Term and soluble T7Prom to create high density immobilized DNA (HDID) in each well ( c ). The beads-immobilized DNA is translated to protein with the addition of cell-free reagents ( d , e ) and the protein is arrayed onto Ni–NTA slides using a microarray printer to produce high density protein microarray slides (HDS) ( f )

Article Snippet: Standard PCR conditions were: 100–150 ng DNA template (pIVEX2.3d-GFP or ompA linearized template, 1 mM MgSO 4 , 1X PCR buffer, 0.3 mM dNTP mix, 0.8 pmol T7Prom soluble primer, 5 μg beads-immobilized primer and 1.25 U Platinum pfx DNA polymerase (Invitrogen) in a final reaction volume of 10 μl.

Techniques: Modification, Amplification, Microarray

Journal: BMC Research Notes

Article Title: Development of repeatable arrays of proteins using immobilized DNA microplate (RAPID-M) technology

doi: 10.1186/s13104-015-1637-3

Figure Lengend Snippet: Western blot analysis of GFP ( a ) and OmpA ( b ) proteins using beads-immobilized DNA template. Proteins were detected using peroxidase-conjugated anti-polyHistidine monoclonal antibody. The calculated protein sizes for GFP and OmpA are 30 and 27 kDa, respectively. DNA templates used for cell-free expression were as follows: a Lane 1 , gfp -SA beads template; lane 2 , gfp -CA beads template; lane 3 , soluble DNA coding for GFP (as positive control); lane 4 , no DNA template (as negative control). b Lane 1 , no DNA template (as negative control); lane 2 , ompA -CA beads template; lane 3 , ompA -SA beads template; lane 4 , soluble ompA protein coding DNA (as positive control)

Article Snippet: Standard PCR conditions were: 100–150 ng DNA template (pIVEX2.3d-GFP or ompA linearized template, 1 mM MgSO 4 , 1X PCR buffer, 0.3 mM dNTP mix, 0.8 pmol T7Prom soluble primer, 5 μg beads-immobilized primer and 1.25 U Platinum pfx DNA polymerase (Invitrogen) in a final reaction volume of 10 μl.

Techniques: Western Blot, Expressing, Positive Control, Negative Control

Journal: BMC Research Notes

Article Title: Development of repeatable arrays of proteins using immobilized DNA microplate (RAPID-M) technology

doi: 10.1186/s13104-015-1637-3

Figure Lengend Snippet: Repetitive cell-free expression of GFP and OmpA proteins using immobilized DNA templates. a Lane 1 , GFP from soluble DNA (as positive control); lanes 2–5 , GFP protein from the first, second, third and fourth sequential rounds of cell-free expression using gfp -CA beads template; lanes 6-9 , GFP protein from the first, second, third and fourth sequential rounds of cell-free expression using gfp -SA beads template; lane 10 , no DNA template (as negative control). b Lanes 1–4 , OmpA protein from the first, second, third and fourth sequential rounds of cell-free expression using ompA -CA beads template; lane 5 , OmpA protein from soluble DNA (as positive control); lane 6 , no DNA template (as negative control)

Article Snippet: Standard PCR conditions were: 100–150 ng DNA template (pIVEX2.3d-GFP or ompA linearized template, 1 mM MgSO 4 , 1X PCR buffer, 0.3 mM dNTP mix, 0.8 pmol T7Prom soluble primer, 5 μg beads-immobilized primer and 1.25 U Platinum pfx DNA polymerase (Invitrogen) in a final reaction volume of 10 μl.

Techniques: Expressing, Positive Control, Negative Control

Journal: BMC Research Notes

Article Title: Development of repeatable arrays of proteins using immobilized DNA microplate (RAPID-M) technology

doi: 10.1186/s13104-015-1637-3

Figure Lengend Snippet: Application of the RAPID-M method for generation of protein arrays. The proteins were printed using the Nano-Plotter™ NP 2.1. Immobilized proteins were in situ purified on Ni–NTA chip and subjected to detection using peroxidase-conjugated anti-polyHistidine antibody followed by addition of TSA-Cy5. Lane 1–4 , proteins from repetitive cycles of first, second, third and fourth rounds of cell-free expression using immobilized DNA template. Proteins are printed in 3 × 3 blocks and each block represents one cycle of cell-free expression using beads-immobilized DNA

Article Snippet: Standard PCR conditions were: 100–150 ng DNA template (pIVEX2.3d-GFP or ompA linearized template, 1 mM MgSO 4 , 1X PCR buffer, 0.3 mM dNTP mix, 0.8 pmol T7Prom soluble primer, 5 μg beads-immobilized primer and 1.25 U Platinum pfx DNA polymerase (Invitrogen) in a final reaction volume of 10 μl.

Techniques: In Situ, Purification, Expressing, Blocking Assay