human jurkat t leukemia cells  (ATCC)

Journal: The Journal of Clinical Investigation

Article Title: MAP4K2 suppresses antitumor immunity in a pancreatic cancer model by promoting Treg differentiation

doi: 10.1172/JCI196379

Figure Lengend Snippet: ( A ) The detected peptide sequences (red color) of the endogenous DDX39B proteins by mass spectrometry–based proteomics analyses using the Myc-tagged MAP4K2 immunocomplexes isolated from Jurkat T cells stimulated with anti-CD3 antibody. ( B ) Reciprocal immunoprecipitation of Flag-tagged MAP4K2 (M4K2) with Myc-tagged DDX39B proteins from lysates of HEK293T cells cotransfected with Flag -MAP4K2 and Myc -DDX39B plasmids. Arrowhead indicates the Flag-tagged DDX39B protein; asterisk indicates heavy-chain signals. ( C ) FRET analysis of HEK293T cells transfected with indicated plasmids encoding CFP-fused MAP4K2 or YFP-fused DDX39B proteins. Means ± SEM are shown. n = 3. ( D ) Confocal microscopy of PLA signals for the interaction between the endogenous MAP4K2 and DDX39B proteins in in vitro differentiated Treg cells. For PLA, red dots represent direct interaction signals. Scale bars: 10 μm. ( E ) Co-IP of the endogenous MAP4K2 and DDX39B proteins from lysates of murine primary T cells stimulated with TGF-β (125 ng/mL). ( F ) Confocal microscopy of CFP-fused MAP4K2 protein, YFP-fused DDX39B protein, FRET, and DAPI in transfected Jurkat T cells. Merge denotes the merge of all 4 color images. Scale bars: 25 μm. * P < 0.05 (2-tailed Student’s t test). Data shown ( B and D – F ) are representative of 3 independent experiments.

Article Snippet: Human Jurkat T leukemia (ATCC, TIB-152) and CRISPR & TARGATT gene editing Jurkat (Jurkat-Cas9, Applied StemCell) cell lines were cultured in RPMI 1640 medium containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Mass Spectrometry, Isolation, Immunoprecipitation, Transfection, Confocal Microscopy, In Vitro, Co-Immunoprecipitation Assay

Journal: The Journal of Clinical Investigation

Article Title: MAP4K2 suppresses antitumor immunity in a pancreatic cancer model by promoting Treg differentiation

doi: 10.1172/JCI196379

Figure Lengend Snippet: ( A ) Deficiency of DDX39B proteins in DDX39B knockout (KO) Jurkat T cells was confirmed by immunoblotting (upper panel). ( B ) Real-time PCR of FOXP3 mRNA levels in WT or DDX39B -KO Jurkat T cells transfected with Myc -MAP4K2 (M4K2 ) plasmid. The mRNA levels of FOXP3 were normalized to GAPDH mRNA levels. Results (mean ± SEM, n = 3) are presented relative to those of vector controls. ( C and D ) Real-time PCR of Foxp3 mRNA levels ( C ) and Foxp3 pre-mRNA (individual junction regions across introns-exons) levels ( D ) in in vitro differentiated Treg cells of T -Map4k2 cKO (cKO) and WT mice. The Foxp3 mRNA levels were normalized to Gapdh mRNA levels ( C ); individual Foxp3 pre-mRNA levels were normalized to total Foxp3 mRNA levels ( D ). Means ± SEM are shown. n = 3. § P < 0.05; (1-tailed Student’s t test); * P < 0.05; ** P < 0.01 (2-tailed Student’s t test).

Article Snippet: Human Jurkat T leukemia (ATCC, TIB-152) and CRISPR & TARGATT gene editing Jurkat (Jurkat-Cas9, Applied StemCell) cell lines were cultured in RPMI 1640 medium containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Knock-Out, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, In Vitro

Journal: The Journal of Clinical Investigation

Article Title: MAP4K2 suppresses antitumor immunity in a pancreatic cancer model by promoting Treg differentiation

doi: 10.1172/JCI196379

Figure Lengend Snippet: ( A ) Real-time PCR of FOXP3 mRNA levels in Jurkat T cells transfected with either Myc -MAP4K2 (M4K2) WT or kinase-dead (K45A) plasmid. The mRNA levels of FOXP3 were normalized to GAPDH mRNA levels. Results (mean ± SEM, n = 3) are presented relative to those of vector controls. ( B ) In vitro kinase assays of purified Myc-tagged MAP4K2 WT or kinase-dead (K45A) mutant proteins, using purified Flag-tagged DDX39B proteins as substrates. Phosphorylation of DDX39B was determined by immunoblotting analyses using anti-pan-phospho-serine (p-Ser) and anti-pan-phospho-threonine (p-Thr) antibodies. ( C ) Mass spectrometry analysis of the tryptic peptides from the DDX39B proteins phosphorylated by MAP4K2 to identify the peptide containing phosphorylated Thr389 or Thr344 residue on DDX39B. ( D ) Real-time PCR of FOXP3 mRNA levels in Jurkat T cells transfected with either Flag- DDX39B WT or phosphomimetic (T344D or T389D) mutant plasmid. The FOXP3 mRNA levels were normalized to GAPDH mRNA levels. Results (mean ± SEM, n = 3) are presented relative to those of vector controls.

Article Snippet: Human Jurkat T leukemia (ATCC, TIB-152) and CRISPR & TARGATT gene editing Jurkat (Jurkat-Cas9, Applied StemCell) cell lines were cultured in RPMI 1640 medium containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, In Vitro, Purification, Mutagenesis, Phospho-proteomics, Western Blot, Mass Spectrometry, Residue

Journal: The Journal of Clinical Investigation

Article Title: MAP4K2 suppresses antitumor immunity in a pancreatic cancer model by promoting Treg differentiation

doi: 10.1172/JCI196379

Figure Lengend Snippet: ( A ) Confocal microscopy of either Flag-tagged DDX39B WT or phosphomimetic (T344D or T389D) mutant proteins in the transfected Jurkat T cells. Nuclei were stained with DAPI. Scale bars: 10 μm. ( B ) Deficiency of MAP4K2 proteins in MAP4K2 knockout (KO) Jurkat T cells was confirmed by immunoblotting ( C ) Real-time PCR of FOXP3 mRNA levels in WT or MAP4K2 KO Jurkat T cells transfected with either Flag- DDX39B WT or phosphomimetic (T389D) mutant plasmid. The mRNA levels of FOXP3 were normalized to GAPDH mRNA levels. Results (mean ± SEM, n = 3) are presented relative to those of vector controls. ( D ) Flow cytometry analyses of the rescue of FOXP3 + cells in T- Map4k2 conditional knockout ( M4k2 cKO) T cells transfected with either GFP- DDX39B WT or phosphomimetic (T389D) mutant plasmid. n = 4. ( E ) Confocal microscopy of CFP-fused MAP4K2, YFP-fused DDX39B, and YFP-fused DDX39B phosphodeficient (T344A or T389A) mutant proteins, as well as DAPI in the transfected Jurkat T cells. Scale bars: 10 μm. ** P < 0.01 (1-way ANOVA and Tukey’s post hoc test). Data shown ( A – E ) are representative of 3 independent experiments.

Article Snippet: Human Jurkat T leukemia (ATCC, TIB-152) and CRISPR & TARGATT gene editing Jurkat (Jurkat-Cas9, Applied StemCell) cell lines were cultured in RPMI 1640 medium containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Confocal Microscopy, Mutagenesis, Transfection, Staining, Knock-Out, Western Blot, Real-time Polymerase Chain Reaction, Plasmid Preparation, Flow Cytometry