Journal: Cell Reports Medicine

Article Title: Tissue-resident memory CAR T cells with stem-like characteristics display enhanced efficacy against solid and liquid tumors

doi: 10.1016/j.xcrm.2023.101053

Figure Lengend Snippet:

Article Snippet: PSMA CAR expression was assessed using a biotinylated recombinant human PSMA protein sourced from Sino Biological, followed by a secondary stain with APC-conjugated streptavidin.

Techniques: Recombinant, CRISPR, Cell Isolation, Staining, Expressing, Software

Journal: Cell Host & Microbe

Article Title: Identification of Human Single-Domain Antibodies against SARS-CoV-2

doi: 10.1016/j.chom.2020.04.023

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Article Snippet: The biotinylated human Tim-3, human IL-33 and human 5T4 were purchased from ACROBiosystems Inc.

Techniques: Virus, Recombinant, Saline, Luciferase, Expressing, Plasmid Preparation, Software

Journal: Traffic (Copenhagen, Denmark)

Article Title: PKC anchoring to GluR4 AMPA receptor subunit modulates PKC-driven receptor phosphorylation and surface expression.

doi: 10.1111/j.1600-0854.2006.00521.x

Figure Lengend Snippet: Figure 1: Effect of PKC on GluR4 phosphory- lation and surface expression in primary cul- tures of retina amacrine-like neurons. A) Effect of PKC activation on GluR4 phosphorylation at Ser842. Cells were incubated with the PKC activator, PMA (200 nM) for 10 min. Cell extracts were prepared, subjected to SDS–PAGE and immunoblotted against phosphorylated GluR4 at Ser842 and total GluR4. The amount of phosphor- ylated GluR4 was normalized to the total amount of GluR4 in each lane. B) Effect of PKC activation on GluR4 surface expression. Biotinylation was performed as described in Materials and Meth- ods. Streptavidin-retained protein complexes were collected and run on SDS–PAGE. Immuno- blot was performed using an antibody against GluR4 C-terminal region. Surface GluR4 was normalized to the total amount of GluR4 in each condition. C, D) Effect of PKC activation on the phosphorylation of surface (C) or intracellular (D) GluR4. All data are expressed as percentage of control and plotted as the mean SEM for the indicated number of experiments performed in independent preparations (*p < 0.05, **p < 0.01, Bonferroni’s test). Representative Western blots are shown.

Article Snippet: EZ-link Sulfo-NHS-SS-biotin, UltraLink Plus Immobilized Streptavidin Gel and the bicinchoninic acid (BCA) protein assay reagent kit were from Pierce (Rockford, IL, USA). pGEX4T-2 vector, glutathioneSepharose 4B, goat polyclonal anti-GST antibody, alkaline-phosphataseconjugated anti-rabbit and anti-mouse secondary antibodies, and the Enhanced Chemifluorescence (ECF) immunodetection substrate were all obtained from Amersham Biosciences (Uppsala, Sweden).

Techniques: Expressing, Activation Assay, Phospho-proteomics, Incubation, SDS Page, Control, Western Blot

Journal: Traffic (Copenhagen, Denmark)

Article Title: PKC anchoring to GluR4 AMPA receptor subunit modulates PKC-driven receptor phosphorylation and surface expression.

doi: 10.1111/j.1600-0854.2006.00521.x

Figure Lengend Snippet: Figure 3: Effect of blocking the PKCg–GluR4 interaction on PKC-driven phosphorylation and plasma membrane expression of GluR4. Cultured HEK 293 cells were transfected with N-terminally Flag-tagged GluR4 or Flag-tagged GluR4AAA, or cotransfected with Flag-tagged GluR4 or Flag-tagged GluR4AAA and PKCg, as indicated. When indicated, cells were stimulated with PMA (200 nM for 10 min). A) Cell extracts were prepared, subjected to SDS–PAGE and immunoblotted against phosphorylated GluR4 at Ser842 and total Flag-GluR4. Phosphorylated GluR4 on Ser842 and total GluR4 were quantified, and the amount of phosphorylated GluR4 was normalized to the total amount of GluR4 in each condition. B) Plasma membrane proteins were biotinylated and purified. Streptavidin-retained protein complexes were collected and run on SDS–PAGE. Immunoblot was performed using an antibody against the Flag epitope. Plasma membrane GluR4 and total GluR4 were quantified, and the amount of surface GluR4 was normalized to the total amount of GluR4 in each condition. All data are expressed as percentage of control and plotted as the mean SEM for the indicated number of experiments performed in independent preparations (*p < 0.05; **p < 0.01, Bonferroni‘s test). Representative Western blots using antibodies against phosphorylated GluR4 at Ser842, surface GluR4 and total GluR4 are shown.

Article Snippet: EZ-link Sulfo-NHS-SS-biotin, UltraLink Plus Immobilized Streptavidin Gel and the bicinchoninic acid (BCA) protein assay reagent kit were from Pierce (Rockford, IL, USA). pGEX4T-2 vector, glutathioneSepharose 4B, goat polyclonal anti-GST antibody, alkaline-phosphataseconjugated anti-rabbit and anti-mouse secondary antibodies, and the Enhanced Chemifluorescence (ECF) immunodetection substrate were all obtained from Amersham Biosciences (Uppsala, Sweden).

Techniques: Blocking Assay, Phospho-proteomics, Clinical Proteomics, Membrane, Expressing, Cell Culture, Transfection, SDS Page, Purification, Western Blot, FLAG-tag, Control

Journal: Science Advances

Article Title: A nonviral, nonintegrating DNA nanovector platform for the safe, rapid, and persistent manufacture of recombinant T cells

doi: 10.1126/sciadv.abf1333

Figure Lengend Snippet: The efficacy of targeting tumor cells expressing the human epitopes CEA, NY-BR1, and CD19 was evaluated in preclinical models. For the CEA model, 1 × 10 6 cells were implanted subcutaneously in NSG mice, and on day 7, the mice were treated with mock, and CAR-T cells were generated with lentivirus and nS/MARt. Tumor growth ( A ) and mice survival ( B ) were monitored. nS/MARt CAR-T cells showed a higher tumor infiltration when compared to Lenti CAR-T cells ( C ), while the human T cells engraftment in both groups appeared similar ( D ). The outgrown tumors showed an antigen loss in the treated groups, while it remains expressed in the control group ( E ). CAR-T cells from the spleens, tumors, and total blood of the treated mice were isolated, and an RCA demonstrated the presence of the episomal plasmids [( F ). M. DNA molecular marker; V, nS/MARt-CEA DNA restriction analysis; +, RCA on purified nS/MARt-CEA subsequentially digested with restriction enzymes]. Similarly, for the NY-BR1 model, cells expressing the epitope were xenografted into NSG mice. The efficacy of tumor killing was recorded as tumor growth ( G ) and survival ( H ). CAR-T cells in the tumors, spleens, and blood of treated mice were found 30 days after injection ( I ), and the episomal maintenance of the vector was demonstrated by RCA ( J ). Nalm-6 cells (1 × 10 5 ) modified to express the luciferase constitutively were injected into NSG mice by tail vein injection. Three days after tumor cell injection, 1 × 10 6 CD19-CAR-T cells generated with mock, nS/MARt, or lentivirus were administered. The tumor growth was monitored through bioluminescent imaging ( K ), as the radiance (p/s/cm 2 /sr), and the statistical analysis was performed with a one-way ANOVA followed by post hoc analysis (** P < 0.001 and *** P < 0.001).

Article Snippet: The expression of the CEA-CAR was detected with the anti-human FCg antibody (polyclonal and R-PE conjugated; Jackson ImmunoResearch), while biotinylated human CD19 (Acro Biosystems) followed by Streptavidin–Alexa Fluor 488 (Life Technologies) was used for the detection of the expression of the CD19-CAR.

Techniques: Expressing, Generated, Control, Isolation, Marker, Purification, Injection, Plasmid Preparation, Modification, Luciferase, Imaging

Journal: Immunity

Article Title: Roquin-2 shares functions with its paralog Roquin-1 in the repression of mRNAs controlling T follicular helper cells and systemic inflammation.

doi: 10.1016/j.immuni.2013.01.011

Figure Lengend Snippet: Figure 1. Roquin-2 Localizes to SGs and P Bodies and Represses Icos mRNA (A) Schematic diagram of Roquin-1 and Roquin-2 proteins and amino acid sequence similarity for each domain. (B and C) HEK293T cells were transfected with V5-RC3H2WT (shown in green), treated with 1 mM sodium arsenite for 1 hr to induce SG formation, and then fixed and stained with anti-eIF3 (B) or anti-DCP1A (C) antibody (red). DNA is stained with 4’,6-diamidino-2-phenylindole (blue). Images in (B) and (C) were all taken at the same magnification, and the scale bar represents 50 mm. (D) Retroviral vectors used to investigate full- length (FL) Icos mRNA (including coding sequence [CDS] and 30UTR) and Icos CDS repression by Roquin-1 and Roquin-2. LTR, long terminal re- peats; J, retroviral packaging element; HuCD4, human CD4 protein; GFP, green fluorescent pro- tein; IRES, internal ribosome entry site. (E) Flow cytometric analysis of NIH 3T3 cells following retroviral cotransduction of Rc3h1 (left panel) or RC3H2 (right panel) with either Icos FL mRNA (top panel) or Icos CDS mRNA (bottom panel). Expression of Roquin-1 or Roquin-2 is assessed by GFP fluorescence, whereas that of ICOS is assessedby human CD4 (HuCD4) reporter. (F) Quantification of HuCD4 (normalized to empty vector) on NIH 3T3 cells cotransduced with Icos FL mRNA and either Rc3h1 or RC3H2. (G) Similar to (F), but NIH 3T3 cells were cotrans- duced with RC3H2 and either Icos FL mRNA or Icos CDS mRNA. The vertical axes in (F) and (G) show the fold difference in HuCD4 expression on NIH 3T3 cells transduced with either Rc3h1 or RC3H2 relative to empty vector. The dashed lines in (F) and (G) indicate full (unrepressed) expression of HuCD4. Data are representative of three inde- pendent experiments. See also Figure S1.

Article Snippet: Antibodies for immunofluorescence studies were as follows: DCP1A (gift from J. Lykke-Andersen, University of California San Diego), eIF3 (Santa Cruz Biotechnology), V5-tag (AbD Serotec), FLAG-tag (Sigma-Aldrich), rabbit antiseraagainstRoquin-2 (gift fromStevenHefeneider; OregonHealth andScience University), andRoquin-1 (NovusBiologicals).

Techniques: Sequencing, Transfection, Staining, Retroviral, Expressing, Plasmid Preparation, Transduction

Journal: Scientific Reports

Article Title: The large extracellular loop of CD63 interacts with gp41 of HIV-1 and is essential for establishing the virological synapse

doi: 10.1038/s41598-021-89523-7

Figure Lengend Snippet: Detection of direct interactions between CD63 and gp41 by FRET. ( A ) Schematic representation of vectors used for the FRET experiments (not to scale), CMV: cytomegalovirus promoter, SP: sequence of murine Ig kappa-chain V-J2-C, NHR: N-terminal heptad repeat of gp41, CHR: C-terminal heptad repeat of gp41, CFP: cyan fluorescent protein, V5 tag: GKPIPNPLLGLDST, CD63: cluster of differentiation 63, YFP: yellow fluorescent protein, FLAG tag: DYKDDDDK, numbers refer to the amino acid sequence. ( B ) Confocal images of HEK293T cells expressing FRET pairs. Areas are marked by frames as regions of interest (ROIs) in red (bleached), green (unbleached) and blue (background) frames. ( C ) Fluorescence intensity ( I ) profiles of CFP (cyan line) and YFP (yellow line) emissions during acceptor photobleaching experiments. Acceptor photobleaching with a 514 nm laser line starts at time point 0 and is repeated at each further time point. ( D ) Quantification of FRET acceptor photobleaching experiments by calculating FRET efficiencies for CFP-YFP (n = 6), CFP/YFP (n = 6), CD63-YFP/SP-gp41-CFP (n = 9), and CD63 C145A,C146A -YFP/SP-gp41-CFP (n = 9). All scale bars: 10 µm.

Article Snippet: We used the following antibodies for PLA experiments: mouse anti-V5 (MCA1360, Serotec), anti-FLAG (NB600–344, NovusBio) and anti-Na + , K + -ATPase (ab98787, Abcam).

Techniques: Sequencing, FLAG-tag, Expressing, Fluorescence

Journal: Scientific Reports

Article Title: The large extracellular loop of CD63 interacts with gp41 of HIV-1 and is essential for establishing the virological synapse

doi: 10.1038/s41598-021-89523-7

Figure Lengend Snippet: Detection of the interaction between CD63 and gp41 by PLA. ( A ) A schematic illustration of direct and indirect PLA experiments. Indirect PLA was performed by detecting FLAG and V5 epitopes by primary antibodies and direct PLA by conjugation of primary antibodies recognizing CD63 and gp41 (2F5) specific epitopes. ( B ) Confocal images of HEK293T cells co-transfected with indicated vectors. Indirect PLA was performed on B1–B4, B9 and direct PLA on B5–B8. ( C ) Quantification of PLA signals per cell obtained for the expressed protein pair SP-gp41-V5/CD63-FLAG from independent transfections: B1 (n = 100), B2 (n = 97), B3 (n = 100), B4 (n = 100), B5 (n = 110), B6 (n = 96), B7 (n = 95), B8 (n = 105), and B9 (n = 160). ( D ) Western blot analysis of transfected HEK293T cells used for PLA experiments expressing CD63-FLAG and SP-gp41-V5; whole-cell lysates were probed with the indicated primary antibodies. ( E ) Confocal images of protein expression control by FLAG and V5 by single recognition PLA. ( F ) Confocal images of indirect immunofluorescent staining for expressed CD63-FLAG and SP-gp41-V5 using transfected HEK293T cells from PLA experiments.

Article Snippet: We used the following antibodies for PLA experiments: mouse anti-V5 (MCA1360, Serotec), anti-FLAG (NB600–344, NovusBio) and anti-Na + , K + -ATPase (ab98787, Abcam).

Techniques: Conjugation Assay, Transfection, Western Blot, Expressing, Staining

Journal: Cell

Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

doi: 10.1016/j.cell.2020.09.033

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Article Snippet: VSV-G antibody , ATCC , Cat # CRL-2700, RRID: AB_2868468.

Techniques: Avidin-Biotin Assay, Virus, Recombinant, FLAG-tag, CtB Assay, Expressing, Plasmid Preparation, Software