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

Article Title: Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts

doi: 10.1073/pnas.1806305115

Figure Lengend Snippet: Next-generation sequencing read counts of gRNA sequences present in the surviving HFFs after AD169 or Merlin (pAL1111) infection

Article Snippet: Libraries A and B of human GeCKO lentiviral gRNA library v2 (lentiCRISPRv2; 1000000048; Addgene) ( 23 , 24 ) were transformed and amplified in Endura electrocompetent cells (60242; Lucigen) following the protocol associated with the library.

Techniques: Next-Generation Sequencing, Infection

Journal: Cell reports

Article Title: Calcineurin is an adaptor required for assembly of the TCR signaling complex

doi: 10.1016/j.celrep.2024.114568

Figure Lengend Snippet: Jurkat cells (A and B) and human T cells (C and D) were treated with CsA at 37°C or transfected with the calcineurin A siRNA cocktail (α and β isoforms) and then activated with soluble anti-CD3 and anti-mouse immunoglobulin G (IgG) for the indicated times. Cell lysates were resolved on SDS-PAGE and blotted with phospho-specific antibodies. The blots were stripped and reblotted for total cellular proteins. The data are representative of 3 independent experiments.

Article Snippet: Two single guiding RNAs (sgRNAs) targeting calcineurin Aα were cloning into pLentiGuide-Puro (Addgene #52963), and two sgRNAs targeting calcineurin Aβ were cloned into pLentiCRISPRv2-neo (Addgene #98292) plasmid using BsmBI restriction sites, as outlined in the protocol from Feng Zhang’s lab (available at Addgene).

Techniques: Transfection, SDS Page

Journal: Cell reports

Article Title: Calcineurin is an adaptor required for assembly of the TCR signaling complex

doi: 10.1016/j.celrep.2024.114568

Figure Lengend Snippet: (A) Unstimulated Jurkat WT and Jurkat CNAαβ-KO cells were lysed, and cell lysates were blotted with anti-pan-calcineurin A (calcineurin Aα and Aβ) antibody. (B) CD3ε expression was detected by flow cytometry. (C) Jurkat WT and Jurkat CNAαβ-KO cells were activated with soluble anti-CD3 and anti-mouse IgG, and cell lysates were immunoblotted for pTCRζ (Tyr83 and Tyr142), pZAP-70 (Tyr319), calcineurin A, and ZAP-70. (D) Quantification of the intensity of the indicated bands with ImageJ software. Data are represented as mean ± SEM. Statistical significance was determined by two-way ANOVA with post hoc tests. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, and ns, not significant.

Article Snippet: Two single guiding RNAs (sgRNAs) targeting calcineurin Aα were cloning into pLentiGuide-Puro (Addgene #52963), and two sgRNAs targeting calcineurin Aβ were cloned into pLentiCRISPRv2-neo (Addgene #98292) plasmid using BsmBI restriction sites, as outlined in the protocol from Feng Zhang’s lab (available at Addgene).

Techniques: Expressing, Flow Cytometry, Software

Journal: Cell reports

Article Title: Calcineurin is an adaptor required for assembly of the TCR signaling complex

doi: 10.1016/j.celrep.2024.114568

Figure Lengend Snippet: (A–C) The indicated Jurkat WT and calcineurin A-deficient cells were dropped onto a stimulatory coverslip (coated with anti-CD3) or non-stimulatory coverslip (coated with anti-CD45) for 5 min prior to fixation. pTCRζ Y142 is shown in red, pLck Y394 is shown in green, and the cell reflection image is shown in gray (scale bar, 10 μm). In each case, a single confocal slice is shown. The pTCRζ Y142 cluster area (B) and co-localization area of pLck Y394 and pTCRζ Y142 (C) in Jurkat WT ( n = 12) and Jurkat CNAαβ-KO cells ( n = 10) were analyzed by Imaris imaging software. The data are representative of 2–3 independent experiments. Data are represented as mean ± SEM. Statistical significance was determined by unpaired t test. ** p < 0.01 and **** p < 0.0001. (D and E) The indicated cells were dropped onto coverslips and analyzed as in (A). pTCRζ Y142 is shown in red and pZAP-70 Y319 (D) or pZAP-70 Y493 (E) is shown in green (scale bar, 10 μm). In each case, a single confocal slice is shown. Co-localization areas were analyzed by Imaris imaging software. Jurkat WT ( n = 22) and Jurkat CNAαβ-KO cells ( n = 13) in (D) and Jurkat WT ( n = 10) and Jurkat CNAαβ-KO cells ( n = 16) in (E) were analyzed. Data are represented as mean ± SEM. Statistical significance was determined by unpaired t test. ** p < 0.05 and **** p < 0.0001. (F–H) Primary human T cells treated with control or calcineurin Aα+Aβ siRNA were dropped onto anti-CD3- or anti-CD45-coated coated coverslips for 5 min prior to fixation. pTCRζ Y142 is shown in red, pLck Y394 is shown in green, and DAPI is shown in blue (scale bar, 10 μm), and in each case, a single confocal slice is shown. The areas of pTCRζ Y142 clusters (G) and pLck Y394 and pTCRζ Y142 co-localization (H) in control ( n = 16) and calcineurin Aα+Aβ knockdown human primary T cells ( n = 17) were quantitated by Imaris imaging software. The data are representative of 2 independent experiments. Data are represented as mean ± SEM. Statistical significance was determined by unpaired t test. * p < 0.05 and ** p < 0.01. (I) CD3ε expression on human primary T cells transfected with control or calcineurin Aα+Aβ siRNA was analyzed by flow cytometry.

Article Snippet: Two single guiding RNAs (sgRNAs) targeting calcineurin Aα were cloning into pLentiGuide-Puro (Addgene #52963), and two sgRNAs targeting calcineurin Aβ were cloned into pLentiCRISPRv2-neo (Addgene #98292) plasmid using BsmBI restriction sites, as outlined in the protocol from Feng Zhang’s lab (available at Addgene).

Techniques: Imaging, Software, Control, Knockdown, Expressing, Transfection, Flow Cytometry

Journal: Cell reports

Article Title: Calcineurin is an adaptor required for assembly of the TCR signaling complex

doi: 10.1016/j.celrep.2024.114568

Figure Lengend Snippet: (A) Jurkat CNAαβ-KO cells stably expressing WT calcineurin Aα or Aβ were analyzed for cell surface CD3ε expression by flow cytometry. (B) Jurkat WT , Jurkat CNAαβ-KO , Jurkat CNAα-WT , and Jurkat CNAβ-WT cells were activated with soluble α-CD3 and anti-mouse IgG for the indicated times. Cell lysates were immunoblotted with pTCRζ (Tyr83 and Tyr142), pZAP-70 (Tyr319), calcineurin A, and ZAP-70. (C) Jurkat WT , Jurkat CNAαβ-KO , Jurkat CNAα-KO , and Jurkat CNAβ-KO cells were stained with anti-CD3ε antibody and analyzed by flow cytometry. (D) Jurkat WT , Jurkat CNAαβ-KO , Jurkat CNAα-KO , and Jurkat CNAβ-KO cells were activated with soluble anti-CD3 and anti-mouse IgG, and cell lysates were immunoblotted for pTCRζ (Tyr83), pZAP-70 (Tyr319), calcineurin A, and ZAP-70.

Article Snippet: Two single guiding RNAs (sgRNAs) targeting calcineurin Aα were cloning into pLentiGuide-Puro (Addgene #52963), and two sgRNAs targeting calcineurin Aβ were cloned into pLentiCRISPRv2-neo (Addgene #98292) plasmid using BsmBI restriction sites, as outlined in the protocol from Feng Zhang’s lab (available at Addgene).

Techniques: Stable Transfection, Expressing, Flow Cytometry, Staining

Journal: Cell reports

Article Title: Calcineurin is an adaptor required for assembly of the TCR signaling complex

doi: 10.1016/j.celrep.2024.114568

Figure Lengend Snippet: (A and B) Jurkat CNAαβ-KO cells were transduced with plasmids encoding WT calcineurin Aα or its inactive mutant (H151A). Images acquired with an ImageStreamX Mark II are representative of unstimulated and anti-CD3-stimulated Jurkat WT , Jurkat CNAαβ-KO , Jurkat CNAα-WT , and Jurkat CNAα-H151A cells after 30 min. DAPI-stained nuclei are shown in blue and NFAT1 is shown in yellow. Quantitative analysis of nuclear translocation was performed with IDEAS image analysis software. Data are represented as mean ± SEM. Statistical significance was determined by two-way ANOVA with post hoc tests. n = 3, **** p < 0.0001, and ns, not significant. (C) Jurkat CNAα-WT and Jurkat CNAα-H151A cells were activated with soluble anti-CD3 and anti-mouse IgG for the indicated times. Cell lysates were immunoblotted with the same antibodies as in . (D) Cells were dropped on stimulatory coverslips for 5 min before fixation. Confocal microscopy was performed to study the formation of microclusters. pTCRζ Y142 is shown in red and pLck Y394 is shown in green. (E and F) Statistical analysis was performed by Imaris imaging software. The pTCRζ Y142 cluster area (E) and co-localization area of pLck Y394 and pTCRζ Y142 (F) were analyzed in Jurkat WT ( n = 12), Jurkat CNAαβ-KO ( n = 10), Jurkat CNAα-WT ( n = 12), and Jurkat CNAα-H151A ( n = 10) cells. Data are represented as mean ± SEM. Statistical significance was determined by one-way ANOVA with post hoc tests. ** p < 0.01 and ns, not significant.

Article Snippet: Two single guiding RNAs (sgRNAs) targeting calcineurin Aα were cloning into pLentiGuide-Puro (Addgene #52963), and two sgRNAs targeting calcineurin Aβ were cloned into pLentiCRISPRv2-neo (Addgene #98292) plasmid using BsmBI restriction sites, as outlined in the protocol from Feng Zhang’s lab (available at Addgene).

Techniques: Transduction, Mutagenesis, Staining, Translocation Assay, Software, Confocal Microscopy, Imaging

Journal: Cell reports

Article Title: Calcineurin is an adaptor required for assembly of the TCR signaling complex

doi: 10.1016/j.celrep.2024.114568

Figure Lengend Snippet: (A and B) Jurkat WT and Jurkat CNAα-KO cells were activated for the indicated times and lysed, and the indicated molecules were immunoprecipitated. Washed immunoprecipitates were separated by SDS-PAGE and analyzed for pTCRζ Y83, RhoH, and ZAP-70. Striped membranes were reprobed with antibodies against the precipitated proteins. The data are representative of 2–3 independent experiments. (C) Jurkat cells expressing RhoH-GFP and calcineurin Aα-mScarlet were plated on coverslips coated with anti-CD3 for 5 min. RhoH is shown in green, calcineurin is shown in red, and pTCRζ Y142 is shown in cyan. (D and E) Jurkat cells were activated for different time points and subjected to immunoprecipitation using protein G-conjugated anti-calcineurin or anti-RhoH antibody. Interaction of signaling protein pTCRζ Y83 was analyzed by immunoblot. Striped membranes were reprobed with anti-pan calcineurin A or anti-RhoH antibody to ensure equal loading of the precipitated complex. Whole-cell lysates from the above were also subjected to immunoblot with the indicated antibody. (F) Jurkat WT ( n = 5) and Jurkat CNAαβ-KO ( n = 4) cells are stimulated with plate-coated α-CD3 for 15 min. PLA signals of calcineurin Aα and RhoH interaction were detected by flow cytometry. Statistical significance was determined by paired t test. * p < 0.05 and ns, not significant.

Article Snippet: Two single guiding RNAs (sgRNAs) targeting calcineurin Aα were cloning into pLentiGuide-Puro (Addgene #52963), and two sgRNAs targeting calcineurin Aβ were cloned into pLentiCRISPRv2-neo (Addgene #98292) plasmid using BsmBI restriction sites, as outlined in the protocol from Feng Zhang’s lab (available at Addgene).

Techniques: Immunoprecipitation, SDS Page, Expressing, Western Blot, Flow Cytometry

Journal: Cell reports

Article Title: Calcineurin is an adaptor required for assembly of the TCR signaling complex

doi: 10.1016/j.celrep.2024.114568

Figure Lengend Snippet:

Article Snippet: Two single guiding RNAs (sgRNAs) targeting calcineurin Aα were cloning into pLentiGuide-Puro (Addgene #52963), and two sgRNAs targeting calcineurin Aβ were cloned into pLentiCRISPRv2-neo (Addgene #98292) plasmid using BsmBI restriction sites, as outlined in the protocol from Feng Zhang’s lab (available at Addgene).

Techniques: Purification, Recombinant, Protease Inhibitor, Cell Recovery, In Situ, Plasmid Preparation, Control, Software, Luminex