13-0289-82

antibody from Invitrogen Antibodies

Targeting: CD28

Provider product page for 13-0289-82

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Product number: 13-0289-82 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD28 Monoclonal Antibody (CD28.2), Biotin, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The CD28.2 monoclonal antibody reacts with the human CD28 molecule, a 44 kDa homodimer expressed by thymocytes, mature T cells and plasma cells. CD28 is a ligand for CD80 (B7-1) and CD86 (B7-2) and is a potent co-stimulator of T cells. Signaling through CD28 augments IL-2 and IL-2 receptor expression as well as cytotoxicity of CD3-activated T cells.
Conjugate: Biotin
Antibody clone number: CD28.2
Concentration: 0.5 mg/mL

CD28 protein structure - 13-0289-82 shown in red.

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Conjugate: Biotin

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Conjugate: Biotin

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Conjugate: Biotin

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Submitted by: Invitrogen Antibodies (provider)
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Conjugate: Biotin

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Conjugate: Biotin

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Conjugate: Biotin

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Conjugate: Biotin

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 CD8 + T cells phenotype in CRC and OVC patients. A . An example of flow cytometric analysis of CD3 + CD8 + T cells analyzed for CD28 and Ki-67, upper panels, and HLA-DR and CD38, lower panels. Percentage of Ki-67 + CD8 T cells in CRC samples (n = 16) B . and OVC samples (n = 22) C . Co-expression of CD38 and HLA-DR on CD8 + T cells in CRC samples (D) and OVC samples (E) .
Conjugate: Biotin

Supportive validation

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Experimental details: Figure 5 ALA-induced PpIX production and PDT of CD4 + CD25 + T cells. Healthy donor PBMCs were either activated in vitro with anti-CD3/IL-2 or anti-CD3/CD28 or kept non-activated (resting) for three days. ( A ) geometric mean fluorescence intensity (Geo. MFI) of PpIX in CD4 + CD25 + cells incubated with 3 mM ALA for 1 h at 37 degC after anti-CD3/IL-2 or anti-CD3/CD28 activation; ( B , C and D ) resting and activated cells were incubated with 3 mM ALA for 1 h and then exposed to the in-house built LED blue light as indicated. The survivals of CD4 + CD25 + T cells were measured before light or 20 h after light exposure with flow cytometry as described in Figure 3 ; ( B ) for resting cells; while ( C and D ) for activated cells with anti-CD3/IL-2 and anti-CD3/CD28, respectively. * p < 0.05.
Conjugate: Biotin

Supportive validation

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Experimental details: FIGURE 4. Rab11Fip5 expression is associated with TNF-alpha production in CD8 + T cells. ( A ) Comparison of expression of genes encoding key immune checkpoints, cytokines, and cytotoxic proteases in CD8 + T cells from immunized RAB11FIP5 -/- mice and immunized control mice, shown as a heat map. TIGIT, LAG3, CTLA4 , and PDCD1 are the genes encoding immune checkpoint markers TIGIT, LAG-3, CTLA-4, and PD-1, respectively. TBX21 is the gene encoding T-Box Transcription Factor 21 (TBX21), also known as T-bet. GZMA, GZMB , and GZMK represent genes encoding granzyme A, B, and K. TNF is the gene encoding the cytokine TNF-alpha. ( B and C ) TNF expression in different cell types from immunized RAB11FIP5 -/- mice or control RAB11FIP5 fl/fl mice shown as fragments per kb of transcript per million mapped reads (FPKM) (B) and relative expression as determined by qPCR in CD8 + T cells (C). ( D and E ) TNF-alpha production by CD8 + T cells correlates with RAB11FIP5 mRNA expression in human PBMC. PBMC from 26 subjects were activated with anti-CD3/CD28 Abs for 3 d and then stimulated with PMA + ionomycin for 5 h before intracellular staining for TNF-alpha. (D) Representative example of intracellular staining for TNF-alpha (gated on CD3 + CD4 - T cells); the TNF-alpha + subset was defined based on a fluorescence minus one control. (E) Pearson analysis of the correlation between the percentage of TNF-alpha + cells within CD8 T cells and RAB11FIP5 mRNA expression in PBMC ( n = 26). Each dot repr
Conjugate: Biotin

Supportive validation

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Experimental details: FIGURE 1 Identification of Tscm CD4 + CD45RA + CD45RO - CD62L + CCR7 + CD127 + CD27 + CD28 + CD95 + CD122 + T (Tscm) cell in human blood and lymph nodes. PBMCs were isolated from the blood of non-small cell lung cancer (NSCLC) patients (n=15) (NSCLC-PBMC) and healthy donors (n=11) (HD-PBMC); lymphocytes were isolated from the tumor-infiltrated lymph node of NSCLC patients who were collected blood at same time (n=7) (NSCLC-Ly); lymphocytes were isolated from the healthy lymph node of non lung cancer patients (n=7) (Normal-Ly), analyzed by flow cytometry. A, Representative flow cytometric analyses of CD4 + CD45RA + CD45RO - CD62L + CCR7 + CD127 + CD27 + CD28 + CD95 + CD122 + T cells, indicating Tscm cells. B, The frequency of the CD4 + Tscm cells in the HD-PBMC, NSCLC-PBMC, Normal-Ly, NSCLC-Ly. The events of CD4 + Tscm cells in the blood and lymph node from NSCLC patients and healthy donors, expressed as the mean+-SEM. C, The frequency of the CD8 + Tscm cells in the HD-PBMC, NSCLC-PBMC, Normal-Ly, NSCLC-Ly, expressed as the mean+-SEM. D, The events of Tscm of CD4 + and CD8 + cells in the blood and the lymph node from NSCLC patients and healthy donors. HD indicates healthy donors; IFN, interferon; PBMC, peripheral blood mononuclear cells; Tscm cell, stem cell-like memory T cell. * P
Conjugate: Biotin

Supportive validation

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Experimental details: Figure 3. Altered FoxO1 gene expression in CD8 + CD28 - T cells. (A) Microarray analysis in CD8 + CD28 + and CD28 - T cells, predicting FoxO1 as an altered transcriptional regulator in CD8 + CD28 - T cells. (B) CCR7 , CD62L , IL7R , and KLRG1 mRNA were assessed by qRT-PCR and normalized to RPL13A mRNA from sorted human T cell populations ( n = 5, paired one-way ANOVA). (C) Peripheral blood mononuclear cells were stained for CD3, CD8, CD28, and indicated markers as in B and analyzed by flow cytometry ( n = 5, paired one-way ANOVA). (D and E) FoxO1 expression in sorted human T cells was measured by Western blot (representative, n = 7, paired two-tailed Student's t test). (F) FoxO1 mRNA was analyzed by qRT-PCR and normalized to RPL13A ( n = 8). (G and H) CD28 + and CD28 - T cells were treated with 20 uM MG132 for 6 h, and FoxO1 expression was measured by Western blot ( n = 3, unpaired two-tailed Student's t test, G shows CD28 - T cells). (I and J) SIRT1 knockdown by Cas9-RNP nucleofection and Western blot for SIRT1 and FoxO1 expression ( n = 9, two-way ANOVA). (K) Nucleofection of recombinant SIRT1 protein into CD8 + T cells and Western blot for SIRT1 and FoxO1 protein 18 h after nucleofection (representative, n = 2). (L) Densitometry of two independent experiments ( n = 2). Data are mean +- SEM of individual donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.000. ns, not significant.
Conjugate: Biotin

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Confirmation of CD19 CAR plasmid into the transfected producer cell line. ( A ) Agarose gel demonstrating cellular mRNA expression (eGFP, CD8a, and CD28) in CD19 CAR plasmid transfected HEK293T cells and control non-transfected cells (WT-control). The beta-actin band is same for each gene of interest because same sample/cDNA was used for PCR amplification of eGFP, CD8a, and CD28. ( B ) Protein expression by flow cytometry (contour plot) demonstrating surface protein expression of CD3z, CD8a, and CD28 co-localized with eGFP expression in CD19 CAR plasmid transfected HEK293T cells (CD19 CAR) and non-transfected cells (WT-control). Bar graph is representation of the three replicates. p -value (*** p < 0.001). This is a representation of cellular CD3z, CD8a, and CD28 expression.
Conjugate: Biotin

Supportive validation

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Experimental details: Figure 5. SIRT1 undergoes lysosomal degradation during aging in mouse and human. a,b, Spleens ( a ) and testes ( b ) from young (2-4 months) and aged (19-26 months) C57BL/6 mice were analyzed by western blotting and RT-qPCR. Data are mean +- s.e.m. ; unpaired two-tailed Students' t-test; n = 4 animals. c,d, Spleens ( c ) and testes ( d ) were analyzed by western blotting and RT-qPCR for SIRT1 expression, from aged (19-24 months) mice subjected to daily i.p. injection of 10 mg/kg Lys05 in PBS or PBS control in 100 muL volume for two weeks. Data are mean +- s.e.m. ; two-tailed Mann-Whitney test. For spleen protein, control group n = 8 animals, Lys05 group n = 7 animals; RNA, control group n = 8 animals, Lys05 group n = 6 animals. For testis protein and RNA, control group n=6 animals, Lys05 group n=6 animals. e. HSPC populations were isolated from young (2-4 months) and aged (20-26 months) C57BL/6 mice, cultured with or without 2 muM Lys05 for 24 hours and analyzed by western blotting and RT-qPCR. For protein, data are mean +- s.e.m. ; one-way ANOVA coupled with Tukey's test; n=6 independent experiments. For RNA, data are mean +- s.e.m. ; unpaired two-tailed Students' t-test; n=4 independent experiments. f. Freshly sorted CD8 + CD28 + (control) and CD8 + CD28 - T cells were treated with Lys05 at doses of 0 and 5 muM for 14 hours, and then were harvested and analyzed by western blotting. Donor age: 53, 54 and 66. Data are mean +- s.d. ; unpaired two-tailed Students' t-test; n = 3
Conjugate: Biotin

Supportive validation

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Experimental details: Fig. 1 Characterization of CD26 expression, mDPP4 enzymatic activity, and cytokine expression in T helper cell lines. (A, B) Jurkat E6 and H9 Th1 cells were harvested and analyzed for the expression of T cell-specific surface markers, including CD3, CD4, and CD26 (A), and for their mDPP4 enzymatic activity (B). (C) H9 Th1 cells were treated with various T cell activators, including T-Activator CD3/CD28, PWM (10 ug/mL), and PMA (10 ug/mL) for 3 h. The expressions of 18 cytokines and 6 MMPs were measured in the culture supernatants of treated cells using two different panels of fluorescent multiplex bead assays. Activated T helper cell-specific cytokines, including IL-2, IL-10, IL-13, GM-CSF, IFN-gamma, and TNF-alpha, are shown. VEGF and MMP 9 are also included as detected high in all the samples. (D-F) H9 Th1 cells were treated with 10 ug/mL PWM for 12 h and subjected to mDPP4 enzymatic assays (D), FACS analysis for T helper cell-specific surface markers such as CD3, CD4, CD26, and CD28 (E), and analysis for apoptosis using an annexin V kit and flow cytometry (F). All analyses were repeated in three independent batches, and the data are shown as the means and standard deviation. ** p
Conjugate: Biotin

Supportive validation

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Experimental details: Figure 3 Expression pattern of CD27 and CD28 on gammadelta T cells in AR. (A) Representative flow cytometry plots of the expression of CD27 and CD28 on gammadelta T cells among PBMCs from HC (n=12) and AR (n=14) subjects. (B-G) Quantification of the percentage of (B) CD27+, (C) CD28+, (D) CD27 + CD28 + , (E) CD27 + CD28 - , (F) CD27 - CD28 + and (G) CD27 - CD28 - gammadelta + T cells in PBMCs. Values are expressed as the mean +- standard deviation. AR, allergic rhinitis; HC, healthy controls; PBMCs, peripheral blood mononuclear cells.
Conjugate: Biotin

Supportive validation

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Experimental details: Figure 2 High Autophagy Level of Intrahepatic T Cells Is Not a Result of a Difference in Differentiation Status or Recent Proliferation (A) Example plot of CD45RA versus CCR7 staining (CD8 + T cells) from a PBMC or IHL sample and summary data for LC3 staining of CD8 + T cell memory subsets (PBMC, 9; and IHL, 15, biological replicates; box whisker, Tukey). (B) Comparison of LC3 staining of CD8 + T cell memory subsets between paired PBMC and IHL samples (9 biological replicates; box whisker, Tukey; outliers shown as dots). (C) Ex vivo CD8 + T cell Ki67 expression. (D and E) Example plots (CD8 + T cells, PBMC) (D) and summary data for LC3 staining on Ki67 - and Ki67 + CD8 + T cells (E) in PBMCs and IHLs ex vivo (10 biological replicates) or after anti-CD3/CD28 stimulation (overnight, 0.5 mug/mL each; three biological replicates) in PBMCs. (F) Histograms showing the dilution of CellTrace Violet (CTV), LC3 staining, and co-staining of LC3 and CTV on CD8 + T cells after 5 days of stimulation with anti-CD3/CD28 (red), compared with that without stimulation (gray) or without CTV staining (black; two representative biological replicates of five, PBMCs). Cells were treated with bafA1 (A-F). Friedman test (ANOVA) with Dunn's post hoc test for pairwise multiple comparisons (A and B). Mann-Whitney t test (C and E). * p < 0.05, ** p < 0.005, *** p < 0.001.
Conjugate: Biotin

Supportive validation

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Experimental details: Figure 4 Enhanced Autophagy Levels Are Linked to Effector Function and Mitochondrial Fitness in Human T Cells (A) Example plots of IFN-gamma, LC3, granzyme B (GzB), and perforin (perf; gated on CD8 + T cells) and histograms of LC3 staining for PBMC ex vivo or after anti-CD3/CD28 stimulation (3 days; see also Figure S3 ). (B) LC3 staining of CD8 + T cells from unstimulated PBMCs (IFN-gamma - ), IFN-gamma - and IFN-gamma + CD8 + T cells after anti-CD3/CD28 stimulation (3 days; eight biological replicates). (C and D) LC3 staining on GzB and perf-expressing CD8 + T cells ex vivo (C) and after anti-CD3/CD28 stimulation (D) (3 days; eight biological replicates; box whisker, Tukey). (E) Example mitochondrial staining of CD8 + T cells in blood (PBMCs; black) and liver (IHLs; red) and summary data for the ex vivo percentage of total CD8 + T cells with depolarized mitochondria (mitoTracker deep red [MtDR] lo ; see also Figure S4 ; PBMCs, 10; and IHLs, 15 biological replicates). (F) Ex vivo percentage of CD8 + T RM cell subsets in the liver with depolarized mitochondria (14 biological replicates; box whisker, Tukey; outliers shown as dots). (G) The percentage of total CD8 + T cells or CD8 + T RM cell subsets with depolarized mitochondria after overnight culture of IHLs with DMSO (untreated), MRT68921 dihydrochloride (10 muM), bafA1 (0.1 muM), or reagent A (chloroquine diphosphate, 1:1000, FlowCellect LC3 kit; 13-15 biological replicates). Cells were treated with bafA1 (A-D). Bars at mea
Conjugate: Biotin

Supportive validation

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Experimental details: Figure 5. Aging and replicative senescence markers in human T lymphocytes. PBMCs from healthy young (< 28 y) and old (> 56 y) donors were stained for CD28 and CD57 and run on LSR II flow cytometer. (A) Autophagy levels (Mean BDS) in CD8 + T cells from young (< 28 y, n = 8) and old (> 56 y, n = 8) donors under basal and basal+I (for 2 h) conditions (mean +- SEM, *p < 0.0499 between young and old basal+I). (B) Representative dot plots from a young and an old donor showing percentages of CD28 and CD57 cells gated on CD8 + T cells. (C) Bar graph showing % of CD8 + lymphocytes with CD28 and CD57 markers in four young and old donors (mean +- SEM, p = 0.0571 for CD8 CD28 population and *p = 0.0286 for CD8 CD57 population). (D) Overlaid histogram of gammaH2AX (DNA double-strand break) levels of CD8 + lymphocytes from three young and old donors gated on CD28 + CD57 - population (geometric mean +- SEM, *p = 0.0286). (E) Overlaid histogram of FAS (CD95) levels of CD8 + lymphocytes from four young and old donors gated on CD28 + CD57 - population (geometric mean +- SEM, *p = 0.0286). PBMCs from four healthy young and old donors were cultured under control and starved conditions for 2 h and stained for CD8, CD57, LC3 and Lyso-ID. (F) Colocalization of LC3 and lysosomal marker in CD8 + CD57 +/- cells, expressed as mean BDS ratio between starved and basal treatments (mean +- SEM, n = 5 (young donors), n = 8 (old donors), **p = 0.0049, *p = 0.035).
Conjugate: Biotin

Supportive validation

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Experimental details: Fig. 5 Bim is required for AICD progression in normal and T-ALL cells. a Expression levels of the indicated proteins in naive hPBT stimulated once, for 3 h (I stim), or twice (II stim) with anti-CD3 (plate-coated) and anti-CD28 antibodies. For the second stimulation, cells have been pre-activated with anti-CD3 (plate-coated) and anti-CD28 antibodies for 48 h and expanded for 6 days in IL2-containing medium. The quantification of the ratio between Bim-L/S protein isoforms and Bcl2 protein is reported for each condition in the graph on the right ( n = 3). b-f AICD has been induced in Jurkat cells silenced for Bim (checked by western blot in B, n = 3). Relative viability (AICD:unstimulated ratio) at 30 h upon AICD induction is indicated in C ( n = 6), western blot analysis of the Opa1 oligomers at 30 h in d ( n = 6; relative quantification on the right), TMRE staining at 26 h by flow cytometry in e ( n = 3; relative quantification on the right), and immunofluorescence analysis of mitochondria morphology (TOM20) and cyt-C localization at 30 h, in single confocal plane, in f ( n = 3; relative quantifications of percentage of cells with fragmented mitochondria, or released cyt-C, on the right). g Expression levels of the indicated genes from T-ALL and healthy bone marrows (HBM) obtained from Leukemia MILE Dataset and analyzed through BloodSpot (HBM: n = 73; T-ALL: n = 174). h Western blot analysis of Bim levels in healthy human Teff cells (stimulated once, and expanded 6 days with
Conjugate: Biotin

Supportive validation

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Experimental details: Fig. 1 Flotillins mediate sorting of internalised TCRzeta into Rab5 and Rab11a endosomes. a Schematic depicting photoactivation to investigate sorting. Top: sorting of photoactivated endosomes (red) into the Rab compartment (green, yellow = merged), bottom: photoactivated endosomes not sorting into the Rab compartment. Dashed lines: quantified compartment mask. b Representative images of photoactivated (dashed region) TCRzeta-PAmCherry sorting into EGFP-Rab4 in WT (left) and FlotKO (right) Jurkat T cells. Cells photoactivated for five frames with 2.5 s intervals, then imaged every 2.5 s for 150 frames. c % endosomal TCRzeta-PAmCherry intensity in Rab4 in WT or FlotKO cells. Dashed line represents timepoint where TCRzeta-PAmCherry intensity significantly diverges between WT and FlotKO. d Percentage reduction in endosomal TCRzeta-PAmCherry intensity in Rab4 from the 50 to 55 s timepoint. Fitted linear regression line is bolded. n.s. indicates slopes are not significantly different. e Representative images of photoactivated (dashed regions) TCRzeta-PAmCherry sorting into EGFP-Rab5 in WT (left) and FlotKO (right) cells. Cells imaged as in b . f Percentage endosomal TCRzeta-PAmCherry in Rab5 in WT or FlotKO cells over time. g Percentage reduction in endosomal TCRzeta-PAmCherry intensity in Rab5 from 50 to 55 s. Fitted linear regression line is bolded. ***Indicates slopes are highly significantly different. h Representative images of photoactivated (dashed region) TCRzeta-PAmCherry
Conjugate: Biotin

Supportive validation

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Experimental details: Fig. 2 TCRzeta recycles from an intracellular compartment labelled by flotillins and Rab11a. a Schematic depicting two-photon photoactivation to measure recycling from intracellular compartments. b Representative confocal images of TCRzeta-PSCFP2 photoactivated with 800 nm light from a two-photon laser, 2.5 um within the cell at the cell centre-localised TCRzeta-mCherry compartment displaying recycling to the plasma membrane over time. Dashed circle = photoactivation region. c % increase in photoactivated TCRzeta-PSCFP2 signal over time at the plasma membrane when activated from cell centre compartments labelled by TCRzeta-mCherry in WT or FlotKO Jurkat T cells or cytoplasmic photoactivation. Greyed region = time of photoactivation. d Mean TCRzeta-PSCFP2 signal from 900 to 1200 s timepoints when photoactivated from the indicated compartments. e Mean intenisty of photoactivated TCRzeta-PSCFP2 2 um within the cell from TCRzeta-mCherry in WT or FlotKO cells. Greyed region = time of photoactivation. f % increase in photoactivated TCRzeta-PSCFP2 signal over time at the plasma membrane when activated from cell centre compartments labelled by mCherry-Rab11a, flotillin1/2-mCherry or TCRzeta-mCherry in WT Jurkat T cells. Grey box = time of photoactivation. Greyed region = time of photoactivation. g Mean TCRzeta-PSCFP2 signal from 900 to 1200 s timepoints when photoactivated from the indicated intracellular compartments. h Percentage of photoactivated TCRzeta-PSCFP2 vesicles within 320
Conjugate: Biotin

Supportive validation

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Experimental details: Fig. 3 Flotillins regulate TCRzeta sorting into Rab11a vesicles for fusion with the plasma membrane. a Representative images of a TCRzeta-GFP vesicle fusion event. Zoomed images below represent the indicated fusion event (white box) at 200 ms intervals. Main image scale bar = 5 um, inset scale bar = 1 um. b TCRzeta-GFP vesicle fusion events per minute in WT and two FlotKO Jurkat T cell lines. c Representative images of TCRzeta-GFP (cyan) vesicle fusion events with either flotillin-1 and flotillin-2 mCherry (magenta, top two panels) or mCherry-Rab11a (magenta, bottom panel). Scale bar = 0.5 um. d Percentage of TCRzeta-GFP fusion events containing either flotillin1/2-mCherry or mCherry-Rab11a. e Representative images displaying a GFP-Rab11a vesicle fusion event. Zoomed images below represent the indicated fusion event (white box) at 200 ms intervals. Main image scale bar = 5 um, inset scale bar = 1 um. f GFP-Rab11a vesicle fusion events per minute in WT and two FlotKO Jurkat T cell lines. g Representative images of TCRzeta-GFP (cyan) vesicle fusion events with mCherry-Rab11a (magenta) in WT Jurkat T cells (top) or fusion with (middle) or without (bottom) mCherry-Rab11a in FlotKO Jurkat T cells. Scale bar = 0.5 um. h Percentage of TCRzeta-GFP fusion events containing mCherry-Rab11a in WT and two FlotKO Jurkat T cell lines. All images captured on a Zeiss ELYRA TIRF microscope. Data points indicate means of independent experiments, error bars indicate mean +- SEM. n.s. = not signi
Conjugate: Biotin

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Flotillins rapidly interact with and maintain the organisation of Rab5 and Rab11a-positive endosomes. a Representative images of flotillin-2-PAmCherry photoactivated at the dashed region failing to interact with EGFP-Rab4 endosomes in WT Jurkat T cells. Cells were photoactivated for five frames with 2.5 s intervals, then imaged every 2.5 s for 60 frames. b Representative images of flotillin-2-PAmCherry photoactivated at the dashed region rapidly interacting with EGFP-Rab5 endosomes in WT Jurkat T cells. Cells were photoactivated and imaged as in a . c Representative images of flotillin-2-PAmCherry photoactivated at the dashed region rapidly interacting with EGFP-Rab11 endosomes in WT Jurkat T cells. Cells were photoactivated and imaged as in a . For a - c zoomed images below are indicated by a white box, main image scale bar = 5 um, inset scale bar = 1 um, and flotillin-2-PAmCherry was co-expressed with untagged flotillin-1. All cells imaged on a Zeiss 880 confocal, photoactivation performed at 405 nm. d Quantification of the percentage of photoactivated flotillin-2-PAmCherry present in Rab4, 5 or 11a. e Representative confocal images of endogenous Rab4, Rab5 and Rab11a detected with corresponding antibodies in WT (top) or FlotKO (bottom) Jurkat T cells. Scale bar = 5 um. f Mean fluorescence dispersion of the indicated markers. Data points indicate mean dispersion from n = 3 biologically independent experiments (Fig. 4a-d, Rab4: 10; 8; 5 cells, Rab5: 10; 12; 10 cells,
Conjugate: Biotin

Supportive validation

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Experimental details: Figure 2 Metformin Ameliorates OXPHOS and Promotes Non-mitochondrial Glycolysis in CD4 + T Cells from O Subjects (A) OCR in a mito stress test assayed by XF of CD4 + T cells following 40 h alphaCD3/alphaCD28 stimulation +- 100 mum MET as indicated. (B) OCR:ECAR ratio calculated by profiles in (A) and Figure S2 C. (C) Relative lactate production after 40-h stimulation per (A). (D) Proton leak calculated from (A) data. (E) MMP measured with TMRE after stimulation per (A). #p = 0.055 versus O. (F) MMP measured following addition of the mitochondrial uncoupler fluoro-carbonyl cyanide phenylhydrazone (FCCP) to unstimulated CD4 + T cells from Y or O subjects. n = 8-10 (A-E) and 12-13 (F). (G) LDH quantification on western blots. Top: representative blot and bottom averages n = 4-6 of group indicated beneath. * p < 0.05 versus Y, #p < 0.05 versus O. Data shown are mean +- SEM. (H) VIP scores, which rank cytokines as most (leftmost) or least (rightmost) important for differentiating overall cytokine profiles between CD4 + T cells from young subjects stimulated +- the LDH inhibitor OA in an orthagonalized model. A VIP score >1 (bracket) is considered important for differentiating inflammatory profiles between groups. All VIP cytokines indicated also differed in post hoc analyses (p < 0.05). Fold change is compared to Y or Y + FCCP. See also Figure S2 .
Conjugate: Biotin

Supportive validation

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Experimental details: Figure 5 Genetic Inhibition of Autophagy Recapitulates Respiratory Profiles of Cells from O Subjects (A) Th17-associated cytokine production by CD4 + T cells from Y subjects, with cells stimulated 40 h alphaCD3/alphaCD28 in the presence of trimetazidine, a fatty acid oxidation inhibitor; alone; or in combination with trehalose, an autophagy activator. n = 4. * p < 0.05 versus Y by one-way ANOVA. (B) Mito stress test XF profiles from 40 h alphaCD3/alphaCD28-stimulated CD4 + T cells from Y or O subjects as indicated. Autophagy dysfunction was induced in cells from Y subjects using siRNA-mediated ATG3 knockdown. n = 8-12. (C and D) OCR:ECAR ratio (C) and ROS generation (D) measured by DCFDA in CD4 + T cells manipulated as indicated. n = 8-12. * p < 0.05 versus Y by SHORE ( Nicholas et al., 2017 ) (B) or one-way ANOVA (C and D). * p < 0.05 versus Y (basal), $p < 0.05 versus Y (max) (C). (E and G) PLSDA analysis differentiated combinatorial ""inflammation"" of CD4 + cells from Y subjects (blue), Y with siRNA-induced autophagy dysfunction (purple), or autophagy dysfunction and metformin (met; orange). n = 8-9. (F and H) VIP scores rank cytokines important for differentiating data clouds in (E) and (G). Comparison of Figure 5 F with 1D highlights profiles that differentiate Y from either O or Y + ATG3 siRNA conditions. n = 8-9. (I) Mitochondrial ROS generation measured by MitoSOX in CD4 + T cells manipulated as indicated; 3 cells/field and 4 fields/slide were imaged using 40x in Zei
Conjugate: Biotin

Supportive validation

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Experimental details: Figure 6 Aging Promotes STAT3 Activation, Mitochondrial Localization, and IL17A/F Promoter Binding (A and B) Quantification of STAT3 ser727 and the mitochondrial complex 1 subunit NDUFA13 localization in CD4 + T cells stimulated for 40 h with alphaCD3/alphaCD28 +- metformin as indicated. n = 4 with multiple field readings shown per N. (C) Phospho (p)-STAT3 T705 expression assayed on western blots as indicated on x axis. n = 6-8. (D) p-STAT3 T705 expression relative to total STAT3 in O cells in the presence of TEMP or MET. n = 4-5. * p < 0.05 versus Y or control siRNA or #p < 0.05 versus O. Fold change is compared with Y or O. (A and D) 3 cells/field and 3-5 fields/slide were imaged using 63x oil immersion in Zeiss microscope. Average fluorescence/field is reported. (E) Chromatin immunoprecipitation assay showing fold-enrichment of p-STAT3 705 on (left) IL-17A or (right) IL-17F promoters. n = 4. All bar graphs show mean +- SEM. (F) Model for parallel metformin-sensitive pathways that drive T cell inflammaging. Activated T cells from O subjects displayed blunted autophagy, which was largely independent of changes in mitochondrial bioenergetics/excess ROS that promote STAT3-mediated activation of a CD4 + T cell inflammaging profile.
Conjugate: Biotin