Journal: Cell reports

Article Title: CRACD Loss Induces Neuroendocrine Cell Plasticity of Lung Adenocarcinoma

doi: 10.1016/j.celrep.2024.114286

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat anti-CD326 (EPCAM) MicroBeads, mouse , Miltenyi Biotec , Cat# 130-105-958; RRID: AB 2936423.

Techniques: Virus, Plasmid Preparation, Recombinant, Modification, cDNA Synthesis, Staining, Cell Recovery, Infection, Transfection, shRNA, Software

Journal: Journal of Biological Chemistry

Article Title: Trans-activators Regulating Neuronal Glucose Transporter Isoform-3 Gene Expression in Mammalian Neurons

doi: 10.1074/jbc.m402735200

Figure Lengend Snippet: FIG. 3. Sp1/Sp3 binds 149- to 124-bp region of the Glut 3 gene. Mobility shift assays were carried out as explained under “Ex- perimental Procedures.” A, mobility shift assay. 32P-Labeled 149- to 124-bp GLUT 3 oligonucleotide (lane 1) was incubated with 5 g of nuclear extracts from N2A cells (lanes 2–4) and 1-day (1d) (lanes 5–7) and 21-day (21d) mouse brain nuclear extracts (lanes 8–10) and sub- jected to electrophoresis followed by autoradiography. The Sp1/Sp3 band shifts are shown. Addition of the anti-Sp1 antibody (lanes 3, 6, and 9) and anti-Sp3 antibody (lanes 4, 7, and 10) caused the supershift as shown. B, Western blot analysis. Presence of Sp1/Sp3 in these nuclear extracts is shown by Western blot analysis. Nuclear extracts (30 g) from N2A cells and 1- and 21-day-old mouse brain were subjected to immunoblot analysis as explained under “Experimental Procedures.” A representative blot showing a 105-kDa Sp1 protein (top panel) and 97- and 60-kDa bands for Sp3 (lower panel) is presented here. C, Sp1/Sp3 binding to 149 to 124 bp of the Glut 3 gene requires post-transla- tional modification. Mouse brain nuclear extracts from 1- and 21-day- old animals were used in mobility shift assays with the Sp1 consensus sequence (Promega) as probe (lanes 1 and 2). Lanes 3–7 contain the 149 to 119 bp (GT3 SP1). Binding reactions carried out using 1- and 21-day-old mouse brain nuclear extracts and human recombinant Sp1 (Promega) are shown in lanes 3–5 in respective order. Mixing experi- ments of the human recombinant Sp1 and nuclear extracts from 1- and 21-day-old mouse brain are shown in lanes 6 and 7.

Article Snippet: One hundred l of pre-cleared chromatin lysate was incubated with 1 g of either Sp3 (Santa Cruz Biotechnology, Santa Cruz, CA), CREB, or pCREB polyclonal antibody (Upstate Biotechnology, Inc.) and shaken on a nutator at 4 °C.

Techniques: Mobility Shift, Labeling, Incubation, Electrophoresis, Autoradiography, Western Blot, Binding Assay, Modification, Sequencing, Recombinant

Journal: Journal of Biological Chemistry

Article Title: Trans-activators Regulating Neuronal Glucose Transporter Isoform-3 Gene Expression in Mammalian Neurons

doi: 10.1074/jbc.m402735200

Figure Lengend Snippet: FIG. 8. Confirmation of Sp3, CREB, and MSY-1 binding to the Glut 3 promoter region. A, ChIP demonstrates that Sp3 binds to the Glut 3 promoter region. PCR amplifications were performed on immunoprecipitated chromatin from N2A cells. PCR amplification product is seen in a 2% agarose gel as a 185-bp DNA fragment detected in the N2A cellular chromatin complexed with proteins that were immunoprecipitated with an antibody (Ab) against Sp3 (lane 3) using primers containing the Sp3-binding region of the Glut 3 gene (192 to 7 bp). Positive controls consisted of 10% of the total chromatin in the absence of immunoprecipitation (lane 2, positive control for PCR) and the mouse GLUT 3 (1553 to 331 bp) gene (control, lane 4). The negative control consisted of no primary antibody but only the secondary antibody (Ab, lane 5). Lane 1, DNA ladder. B, ChIP assay demonstrates that CREB/pCREB binds to the Glut 3 promoter region. PCR amplifications were performed on immunoprecipitated chromatin from N2A cells. PCR amplification product is seen in a 2% agarose gel as a 185-bp DNA fragment detected in the N2A cellular chromatin complexed with proteins that were immunoprecipitated with antibodies against either CREB (lane 4) or pCREB (lane 5) using primers containing the CREB binding region of the Glut 3 gene (192 to 7 bp). Negative controls consisted of dialysis buffer with no chromatin (Chromatin, lane 2), no primary antibody but staphylococcus A lysate (Ab, lane 3), whereas the positive control consisted of 10% of the total chromatin in the absence of immunoprecipitation (lane 6, positive control for PCR). Lane 1, DNA ladder. C, ChIP assay demonstrates no CREB/pCREB binding to the Glut 3 promoter in COS-7 cells. PCR amplification product is seen in the 2% agarose gel as a 185-bp DNA fragment detected only in the positive DNA control (control) which consisted of the mouse GLUT 3 (1553 to 331 bp) gene (lane 2, positive control for PCR), whereas no amplification product was noted in chromatin obtained from COS-7 (monkey kidney fibroblasts, which served as a negative cellular control) cells and immunoprecipitated with either the CREB (lane 3) or the pCREB (lane 4) antibodies or in 10% of the total chromatin in the absence of immunoprecipitation (lane 5). Lane 1, DNA ladder. D, ChIP assay demonstrates that MSY-1 binds to the Glut 3 promoter region. A 2% agarose gel demonstrates PCR amplifications that were performed on immunoprecipitated chromatin from N2A cells. PCRs targeted at amplifying DNA containing the MSY-1-binding site on the Glut 3 gene (89 to 260 bp) from immunoprecipitated protein complexed to chromatin in the presence of an anti-MSY-1 antibody (MSY-1 Ab, lane 4) revealed a 354-bp region. Negative control consisted of no primary antibody for MSY-1 but only the secondary antibody (Ab, lane 5). Positive controls consisted of 10% of the total chromatin (input, lane 3) and the mouse Glut 3 (1553 to 331 bp) gene (control, lane 2). Lane 1, DNA ladder. E, ChIP assay to demonstrate an interaction between CREB and MSY-1. PCR amplifications were performed on immunoprecipitated chromatin from N2A cells. A 2% agarose gel demonstrating PCR-amplified products containing the CREB and MSY-1 binding domains (185 bp) (lanes 2, 4, and 6) and only the MSY-1 binding domain (354 bp) (lanes 3, 5, and 7) region of the Glut 3 gene obtained by immunoprecipitating proteins complexed to chromatin with either the CREB antibody (lanes 2 and 3) and the MSY-1 antibody (lanes 4 and 5) but not with the IgG which served as the negative antibody control (lanes 6 and 7). Lane 1, DNA ladder. A 185-bp amplification product is seen when the CREB and MSY-1-binding sites containing region of the Glut 3 gene (192 to 7 bp) was amplified from chromatin that was immunoprecipitated with either the CREB (lane 2) or MSY-1 antibodies (lane 4). a 354-bp amplification product is seen when only the MSY-1-binding site containing region of the Glut 3 gene (89 to 260 bp) was amplified from chromatin that was immunoprecipitated with the MSY-1 antibody (lane 5) but not when the CREB antibody was used (lane 3). E, co-immunoprecipitation assay demonstrates an interaction between MSY-1 and CREB. Chromatin from N2A cells was immunoprecipitated with the MSY-1 antibody (lane 2), and this immunoprecipitated antibody-antigen-DNA complex and HeLa cell nuclear extract (lane 1, positive control) were subjected to Western blot analysis employing the CREB antibody (1:1000) which demonstrated an 43-kDa band.

Article Snippet: One hundred l of pre-cleared chromatin lysate was incubated with 1 g of either Sp3 (Santa Cruz Biotechnology, Santa Cruz, CA), CREB, or pCREB polyclonal antibody (Upstate Biotechnology, Inc.) and shaken on a nutator at 4 °C.

Techniques: Binding Assay, Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Positive Control, Control, Negative Control, Co-Immunoprecipitation Assay, Western Blot

Journal: Immunology

Article Title: CD8 + T-cell recognition of a synthetic epitope formed by t -butyl modification

doi: 10.1111/imm.12398

Figure Lengend Snippet: T-cell responses against Bax peptides and mapping the specificity of the CD8 + T-cell clone 6C5. (a) Purified CD8 + T cells were cultured with irradiated autologous activated B-CLL cells and Bax peptides 601–23 for 5 weeks before testing by interferon- γ (IFN- γ ) ELISpot. Antigen-presenting cells (APC) were autologous activation B-CLL cells. Numbers shown are spots/10 5 T cells (mean of triplicates ± SD, n = 1) Statistical analysis (unpaired two-tailed t -test) was carried out using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). (b) T-cell cultures generated by limited dilution were tested for the recognition of Bax peptides 601–23 by IFN- γ ELISpot. T cells were plated (∼2 × 10 4 to 3 × 10 4 /well) with APC or with APC + peptides at a 1 : 1 ratio. APC were T2 cells. Background responses (T cells + T2) were subtracted from the data ( n = 1). (c) 6C5 was tested by IFN- γ ELISpot against the Bax peptide pool 601–23, split pools, and individual peptides. T cells were plated (1 × 10 4 /well) in triplicate with T2 or T2 + peptides at a 1 : 1 ratio. Background response (T cells + T2) was subtracted from the data (5 SFC/10 4 cells) (mean ± SD of triplicates, n = 2). (d) 6C5 was assayed against T2 cells pulsed with varying concentrations (20–6·25 μg/ml) of Bax P603 and Bax P605 at 1 : 1 ratio for 18 hr. Cell-free supernatants were harvested and tested for the presence of IFN- γ by ELISA. The EC 50 value was calculated using the fitted curve, P603 – 4·92 µ m and P605 > 100 µ m (mean ± SD of duplicates, n = 3).

Article Snippet: CD8 + T cells were immunomagnetically enriched from peripheral blood mononuclear cells using anti-human CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Woking, UK).

Techniques: Purification, Cell Culture, Irradiation, Enzyme-linked Immunospot, Activation Assay, Two Tailed Test, Software, Generated, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: CD8 + T-cell recognition of a synthetic epitope formed by t -butyl modification

doi: 10.1111/imm.12398

Figure Lengend Snippet: 6C5 recognition of crude P603 but not highly purified P603. Representative sample of flow cytometry analysis (three independent experiments) of intracellular staining of interferon- γ (IFN- γ ). T cells (1 × 10 5 /tube) were cultured in the presence of T2 or T2 + Bax peptide P603 (> 95% Pure, 10 μg/ml) or P603 (77% Pure, 10 μg/ml) at a 1 : 1 ratio for 5 hr. Lymphocytes were gated based on their forward and side scatter profile and then doublet exclusion was performed based on forward scatter height versus forward scatter width. T cells were then gated on CD3 + CD8 + cells and IFN- γ production was assessed through intracellular staining with anti-IFN- γ (mean ± SD of duplicates, n = 3). Statistical analysis (unpaired two-tailed t -test) was carried out using GraphPad Prism .

Article Snippet: CD8 + T cells were immunomagnetically enriched from peripheral blood mononuclear cells using anti-human CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Woking, UK).

Techniques: Purification, Flow Cytometry, Staining, Cell Culture, Two Tailed Test

Journal: Immunology

Article Title: CD8 + T-cell recognition of a synthetic epitope formed by t -butyl modification

doi: 10.1111/imm.12398

Figure Lengend Snippet: 6C5 recognizes a peptide present in fraction 8. (a) CD8 + T-cell clone 6C5 was cultured in the presences of various peptide preparations representative of potential C-terminal (GTPT) and N-terminal (LSYF) deletions, as well as C-terminal additions. T cells (1 × 10 5 /tube) were cultured in the presence of T2 or T2 + peptides at a 1 : 1 ratio for 5 hr. Interferon- γ (IFN- γ ) production was assessed through intracellular staining with anti-IFN- γ . To facilitate gating, the cultures were also co-stained with anti-CD3 and anti-CD8. Background (T-cells + T2) were subtracted from the data (mean ± SD, n = 3). (b) Fractionation was performed by HPLC (Waters 2525 pump and 2996 detector) with a Vydac 218TP 250 × 22 mm column. Peptides were eluted with a binary gradient of 0 min 95% A, 1 min 95% A, 16 min 50% A where solvent A = H 2 O, 0·05% trifluoroacetic acid (TFA) and solvent B = acetonitrile, 0·05% TFA at a flow rate of 22·9 ml/min. The chromatogram shows the absorbance at 220 nm. (c) CD8 + T-cell clone 6C5 was cultured in the presence of fractions (F1–F8) generated from the fractionation of the P1 peptide preparation (crude full length 9mer, LLYSFGTPT). T cells (1 × 10 5 /tube) were cultured in the presence of T2 or T2 + peptides at a 1 : 1 ratio for 5 hr. Lymphocytes were gated based on their forward and side scatter profile and then doublet exclusion was performed based on forward scatter height versus forward scatter width. T cells were then gated on CD3 + CD8 + cells and IFN- γ production was assessed through intracellular staining with anti-IFN- γ . Background (T-cells + T2) was subtracted from the data (mean ± SD, n = 3). Statistical analysis (unpaired two-tailed t -test, in comparison to P603 > 95% Pure) was carried out using GraphPad Prism . Significance is indicated by ****< 0·0001, ***0·0001–0·001, **0·001–0·01 and *0·01–0·05.

Article Snippet: CD8 + T cells were immunomagnetically enriched from peripheral blood mononuclear cells using anti-human CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Woking, UK).

Techniques: Cell Culture, Staining, Fractionation, Solvent, Generated, Two Tailed Test, Comparison

Journal: Immunology

Article Title: CD8 + T-cell recognition of a synthetic epitope formed by t -butyl modification

doi: 10.1111/imm.12398

Figure Lengend Snippet: Butylation of the tyrosine residue of p603 confers peptide reactivity. (a) Structure of modified peptide containing the alkylated residue Tyr(3- t Bu). Unmodified P603 (> 95% Pure) was reacted with methylpropene and trifluoroacetic acid (TFA), or Boc 2 O and TFA to induce t Bu groups on the tyrosine residue. (b) T cells (1 × 10 5 /tube) were cultured in the presence of T2 or T2 + P603 (> 95% Pure, 10 μg/ml), P603 (77% Pure, 10 μg/ml) or the two P603 modified peptides (di- tert -butyl dicarbonate and methylpropene) at a 1 : 1 ratio for 5 hr. Lymphocytes were gated based on their forward and side scatter profile and then doublet exclusion was performed based on forward scatter height versus forward scatter width. T cells were then gated on CD3 + CD8 + cells and IFN- γ production was assessed through intracellular staining with anti-IFN- γ . Background (T-cells + T2) were subtracted from the data (mean ± SD, n = 3). Statistical analysis (unpaired two-tailed t -test, in comparison to P603 > 95% Pure) was carried out using Graphpad Prism . Significance was indicated by ****< 0·0001.

Article Snippet: CD8 + T cells were immunomagnetically enriched from peripheral blood mononuclear cells using anti-human CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Woking, UK).

Techniques: Residue, Modification, Cell Culture, Staining, Two Tailed Test, Comparison

Journal: Immunology

Article Title: CD8 + T-cell recognition of a synthetic epitope formed by t -butyl modification

doi: 10.1111/imm.12398

Figure Lengend Snippet: Activation of 6C5 is induced by t Bu P603. (a) and (b) Representative sample of flow cytometry analysis (three independent experiments) of intracellular staining of interferon- γ (IFN- γ ) and surface staining of CD107 α , respectively. T cells (1 × 10 5 /tube) were cultured in the presence of T2 or T2 + P603 (> 95% Pure, 10 μg/ml), P603 (77% Pure, 10 μg/ml) or P603 t Bu (> 98% Pure, 10 μg/ml) at a 1 : 1 ratio for 5 hr. IFN- γ production was assessed through intracellular staining with anti-IFN- γ and CD107 α expression through surface staining with anti-CD107 α . To facilitate gating, the cultures were also co-stained with anti-CD3 and anti-CD8 (mean ± SD of duplicates, n = 3). Statistical analysis (unpaired two-tailed t -test) was carried out using GraphPad Prism . (c) 5 × 10 5 T2 cells were pulsed with varying concentrations of P603 (77% Pure, 10 μg/ml to 1 × 10 −6 μg/ml) and P603 t Bu (> 98% Pure, 10 μg/ml to 1 × 10 −6 μg/ml). After 1 hr, unbound peptide was washed off and the pulsed T2 cells were cultured with 1 × 10 5 6C5 CD8 + T cells at a 1 : 1 ratio. Cell-free supernatant was harvested and tested for the presence of IFN- γ by ELISA (mean ± SD, n = 3). (d) 1 × 10 5 6C5 CD8 + T-cells were cultured at 1 : 1 ratio with T2 cells in the presence of varying concentrations of P603 (77% Pure, 10 μg/ml to 1 × 10 −6 μg/ml) and P603 t Bu (> 98% Pure, 10 μg/ml to 1 × 10 −6 μg/ml) for 5 hr. Lymphocytes were gated based on their forward and side scatter profile and then doublet exclusion was performed based on forward scatter height versus forward scatter width. T cells were then gated on CD3 + CD8 + cells and changes in the surface expression of CD107 α were determined through culturing the cells in the presences of anti-CD107 α (mean ± SD, n = 3). The EC 50 value was calculated using the fitted curve using Graphpad Prism .

Article Snippet: CD8 + T cells were immunomagnetically enriched from peripheral blood mononuclear cells using anti-human CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Woking, UK).

Techniques: Activation Assay, Flow Cytometry, Staining, Cell Culture, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: T-bet+ CXCR3+ B cells drive hyperreactive B-T cell interactions in multiple sclerosis

doi: 10.1016/j.xcrm.2025.102027

Figure Lengend Snippet: AP results in BTEC formation, which is BTK dependent (A and B) Staining of B cells (CD19 and CD20) and T helper cells (CD3 and CD4) after 7 days of (A) anti-IgM stimulation or (B) AP in PBMCs (natalizumab-treated RRMS = NAT) by flow cytometry (top panel) and by 4i (bottom panel). Nuclei were labeled with DAPI (blue). Images show increasing magnification from left to right, 49-(well), 4-, and 1-site view. CFSE stands for carboxy fluorescein succinimidyl ester. Scale bar: 200 μm. (C) AP response upon vehicle (top) or BTK inhibitor (BTKi) treatment (bottom) assessed by flow cytometry (left) and 4i (right) including staining for pERK (cyan), pAKT (green), and PCNA (red). Representative image from one donor (NAT, n = 3). Scale bar: 50 μm. (D) 2D projection of local morphology centroids, colored by the mean fluorescence intensity (MFI) values of PCNA, without (AP) or with BTKi treatment to visualize the spatial intensity distribution of cell proliferation. (E) Computation of nearest neighbor statistics using G(r) function on local morphology centroids, for AP upon vehicle or BTKi treatment to measure deviations from complete spatial randomness in favor of clustering or regular patterns. G(r) values above or below the simulation envelope imply clustering or dispersion, respectively. (F) Representative 4i image of BTECs following AP ( n = 3 NAT). Images on the right show magnified 1-site view. Scale bar: 50 μm. (G) Top: 2D projection of cell morphology centroids, colored by B cells (green), T cells (red), and unclassified (gray). Bottom: G(r) function computed on cell morphology centroids based on (F). See also Figure S1 and Table S1 .

Article Snippet: CD19 MicroBeads, human , Miltenyi Biotec , Cat# 130-050-301.

Techniques: Staining, Flow Cytometry, Labeling, Fluorescence, Dispersion

Journal: Cell Reports Medicine

Article Title: T-bet+ CXCR3+ B cells drive hyperreactive B-T cell interactions in multiple sclerosis

doi: 10.1016/j.xcrm.2025.102027

Figure Lengend Snippet:

Article Snippet: CD19 MicroBeads, human , Miltenyi Biotec , Cat# 130-050-301.

Techniques: In Vitro, Control, Recombinant, Staining, Purification, Electron Microscopy, Lysis, Ubiquitin Proteomics, Cell Isolation, Enzyme-linked Immunosorbent Assay, Isolation, Multiplex Assay, Quantitation Assay, Modification, Library Quantification, RNA Sequencing, Sequencing, Software, Cell Culture, Sterility

Journal: STAR Protocols

Article Title: Protocol for Efficient CRISPR/Cas9/AAV-Mediated Homologous Recombination in Mouse Hematopoietic Stem and Progenitor Cells

doi: 10.1016/j.xpro.2020.100028

Figure Lengend Snippet: FACS Plots Showing Lin − Sca1 + c-Kit + (LSK) Cell Fractions after MACS Purification and 2 Days of Culture Bone marrow cells were stained with α-mouse lineage cocktail, Sca1, and c-Kit antibodies. (A) The negative fraction of the LS column should be almost completely depleted of Sca1 + cells. (B) The positive fraction eluted from the LS or MS separation column is enriched in LSK cells and progenitor cells, which all express Sca1. The LSK population should comprise 6 to 8% of the Sca1 + cell fraction. Lymphoid progenitor cells require IL-7 to survive and will be depleted upon culturing with 50 ng/mL mTPO, mSCF, mFLT-3, and hIL-11. (C) Purified LSK cell fraction after 2 days of culture in HSPC medium.

Article Snippet: Anti-mouse Sca1 MicroBead Kit (VioBrightTM FITC) , Miltenyi Biotec , Cat#130-123-124.

Techniques: Purification, Staining

Journal: STAR Protocols

Article Title: Protocol for Efficient CRISPR/Cas9/AAV-Mediated Homologous Recombination in Mouse Hematopoietic Stem and Progenitor Cells

doi: 10.1016/j.xpro.2020.100028

Figure Lengend Snippet:

Article Snippet: Anti-mouse Sca1 MicroBead Kit (VioBrightTM FITC) , Miltenyi Biotec , Cat#130-123-124.

Techniques: Virus, Recombinant, DNA Extraction, Cloning, Modification, Plasmid Preparation, Software, Adhesive, Real-time Polymerase Chain Reaction, Sterility, Centrifugation