rabbit anti phospho stat5  (Bioss)

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: ( A ) OT1 lymph node cell suspensions were SIINFEKL peptide activated for 36 hr, washed then cultured with no cytokine, IL-15 (20 ng/mL) or IL-2 (20 ng/mL) for 4 or 24 hr. Western blots of PIM1 (two isoforms of 44 and 34 kDa, non-specific band indicated by *), PIM2 (three isoforms of 40, 37, and 34 kDa) or pSTAT5 Y694 expression. ( B ) Schematic of cytokine driven memory and effector CD8 T cell expansion and differentiation cultures. Lymph node or spleen cell suspensions were activated for 2 days with TCR stimulus + cytokine, washed, then split daily into fresh media + cytokine. ( C ) WT (Ly5.1) and Pim dKO LN suspensions were mixed at a 50:50 ratio for T cells and cultured as outlined in ( B ) with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), and CD8 T cell number was measured daily. ( D ) WT and Pim dKO T cells were expanded with IL-15 in separate cultures as per ( B, C ) and % live cells (PI-ve) were assessed on days 4 and 6 (two-way ANOVA). ( E–J ) WT and Pim dKO CD8 T cells were activated with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), expanded with IL-15 as per ( B ), with an additional CD4 T cell magnetic depletion step on day 3 of culture. CD8 T cells were harvested on day 6 for parallel RNAseq and proteomic analysis. An interactive version of the proteomics expression data is available for exploration on the Immunological Proteome Resource website: immpres.co.uk ( E ) Fold-change in mRNA expression between Pim dKO and WT versus average mRNA expression (TPM). mRNA expression (Transcripts per million, TPM) of ( F ) secondary lymphoid homing receptors Sell, Ccr7, S1pr1 and ( G ) key transcription factors involved in CD8 T cell memory differentiation and maintenance Tcf7, Klf2, Foxo1, Foxo3, Id3. ( H ) WT vs Pim dKO protein copy numbers, differentially expression proteins (FC >1.5, q<0.05) are highlighted in red ( I ) Protein copy numbers per cell for key mitochondrial proteins DRP1, OPA1, and CPT1A. ( J ) WT vs Pim dKO protein copy numbers, mitochondrial proteins (as defined in MitoCarta 3.0) are highlight in pink. Symbols in bar charts represent biological replicates, symbols in ( C, E, H, J ) represent the mean. Error bars show mean ± S.D. Data are representative of ( A ) n=3 or show pooled data from ( C ) n=4, and ( D ) n=5 biological replicates with data collected over at least two independent experiments. Quantitative proteomics and RNAseq was performed on biological triplicates. ** q≤0.01, fold-change (FC) shown on bar graphs when q<0.05. Figure 2—source data 1. PDF files containing labelled and uncropped images for western blots displayed in . Figure 2—source data 2. Original files for western blot images displayed in . Figure 2—source data 3. Raw values plotted in .

Article Snippet: Cells were stained at 1:200 with rabbit anti-phospho STAT5 (Y694) (C11C5, Cell Signaling Technologies cat#9359 S) followed by anti-rabbit IgG Fab2 Alexa Fluor 647 (Cell Signaling Technologies cat#4414 S).

Techniques: Cell Culture, Western Blot, Expressing, Quantitative Proteomics

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: ( A ) Estimated copies per cell of PIM1 and PIM2 protein from published quantitative proteomics analysis ; of CD8 T cells expanded in IL-2 or IL-15 as outlined in . ( B–D, F, G ) WT (Ly5.1) and Pim dKO lymph node or spleen single-cell suspensions were mixed at a 50:50 ratio of T cells, activated for 2 days with αCD3/αCD28 (both 0.5 µg/mL) and IL-2 (20 ng/mL), washed then split into fresh medium containing IL-2 (20 ng/mL) daily (as per ). Some of the mixed cell suspensions were also cultured in IL-7 (5 ng/mL) to sustain a naive T cell reference. ( B ) WT and Pim dKO CTL were treated 1 hr +/- Jak1/3 inhibitor Tofacitinib (100 nM; negative control) before pSTAT5 Y694 expression was measured on day 3 and 6 of culture, ( C ) surface CD25 expression was measured on days 3 and 6 of culture, ( D ) CD8 T cell number vs time was calculated, ( F ) CD8 T cell FSC-A, SSC-A and surface activation markers (CD44, CD71) were measured on days 3 and 6 of culture ( G ) expression of adhesion molecule CD62L was measured daily. ( E ) WT and Pim dKO T cells were activated and expanded with IL-2 as per ( B-D ) and ( F, G ) except in separate cultures and % live cells (PI-ve) was assessed on days 4 and 6 (two-way ANOVA). Symbols in bar charts represent biological replicates, symbols in ( D ) represent the mean. Error bars show mean ± S.D. Data are representative of ( B, G ) n=4, ( C, F ) n=6 or show pooled data from ( D ) n=4, ( E ) n=6 biological replicates with data collected over at least two independent experiments. Figure 3—source data 1. Raw values plotted in .

Article Snippet: Cells were stained at 1:200 with rabbit anti-phospho STAT5 (Y694) (C11C5, Cell Signaling Technologies cat#9359 S) followed by anti-rabbit IgG Fab2 Alexa Fluor 647 (Cell Signaling Technologies cat#4414 S).

Techniques: Quantitative Proteomics, Cell Culture, Negative Control, Expressing, Activation Assay

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: ( A ) OT1 lymph node cell suspensions were SIINFEKL peptide activated for 36 hr, washed then cultured with no cytokine, IL-15 (20 ng/mL) or IL-2 (20 ng/mL) for 4 or 24 hr. Western blots of PIM1 (two isoforms of 44 and 34 kDa, non-specific band indicated by *), PIM2 (three isoforms of 40, 37, and 34 kDa) or pSTAT5 Y694 expression. ( B ) Schematic of cytokine driven memory and effector CD8 T cell expansion and differentiation cultures. Lymph node or spleen cell suspensions were activated for 2 days with TCR stimulus + cytokine, washed, then split daily into fresh media + cytokine. ( C ) WT (Ly5.1) and Pim dKO LN suspensions were mixed at a 50:50 ratio for T cells and cultured as outlined in ( B ) with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), and CD8 T cell number was measured daily. ( D ) WT and Pim dKO T cells were expanded with IL-15 in separate cultures as per ( B, C ) and % live cells (PI-ve) were assessed on days 4 and 6 (two-way ANOVA). ( E–J ) WT and Pim dKO CD8 T cells were activated with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), expanded with IL-15 as per ( B ), with an additional CD4 T cell magnetic depletion step on day 3 of culture. CD8 T cells were harvested on day 6 for parallel RNAseq and proteomic analysis. An interactive version of the proteomics expression data is available for exploration on the Immunological Proteome Resource website: immpres.co.uk ( E ) Fold-change in mRNA expression between Pim dKO and WT versus average mRNA expression (TPM). mRNA expression (Transcripts per million, TPM) of ( F ) secondary lymphoid homing receptors Sell, Ccr7, S1pr1 and ( G ) key transcription factors involved in CD8 T cell memory differentiation and maintenance Tcf7, Klf2, Foxo1, Foxo3, Id3. ( H ) WT vs Pim dKO protein copy numbers, differentially expression proteins (FC >1.5, q<0.05) are highlighted in red ( I ) Protein copy numbers per cell for key mitochondrial proteins DRP1, OPA1, and CPT1A. ( J ) WT vs Pim dKO protein copy numbers, mitochondrial proteins (as defined in MitoCarta 3.0) are highlight in pink. Symbols in bar charts represent biological replicates, symbols in ( C, E, H, J ) represent the mean. Error bars show mean ± S.D. Data are representative of ( A ) n=3 or show pooled data from ( C ) n=4, and ( D ) n=5 biological replicates with data collected over at least two independent experiments. Quantitative proteomics and RNAseq was performed on biological triplicates. ** q≤0.01, fold-change (FC) shown on bar graphs when q<0.05. Figure 2—source data 1. PDF files containing labelled and uncropped images for western blots displayed in . Figure 2—source data 2. Original files for western blot images displayed in . Figure 2—source data 3. Raw values plotted in .

Article Snippet: Antibody , anti-phospho STAT5 Y694 (Rabbit, monoclonal, C11C5) , Cell Signaling Technology , Cat # 9359 S, RRID: AB_823649 , intracellular stain 1:200; western blot 1:1000.

Techniques: Cell Culture, Western Blot, Expressing, Quantitative Proteomics

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: ( A ) Estimated copies per cell of PIM1 and PIM2 protein from published quantitative proteomics analysis ; of CD8 T cells expanded in IL-2 or IL-15 as outlined in . ( B–D, F, G ) WT (Ly5.1) and Pim dKO lymph node or spleen single-cell suspensions were mixed at a 50:50 ratio of T cells, activated for 2 days with αCD3/αCD28 (both 0.5 µg/mL) and IL-2 (20 ng/mL), washed then split into fresh medium containing IL-2 (20 ng/mL) daily (as per ). Some of the mixed cell suspensions were also cultured in IL-7 (5 ng/mL) to sustain a naive T cell reference. ( B ) WT and Pim dKO CTL were treated 1 hr +/- Jak1/3 inhibitor Tofacitinib (100 nM; negative control) before pSTAT5 Y694 expression was measured on day 3 and 6 of culture, ( C ) surface CD25 expression was measured on days 3 and 6 of culture, ( D ) CD8 T cell number vs time was calculated, ( F ) CD8 T cell FSC-A, SSC-A and surface activation markers (CD44, CD71) were measured on days 3 and 6 of culture ( G ) expression of adhesion molecule CD62L was measured daily. ( E ) WT and Pim dKO T cells were activated and expanded with IL-2 as per ( B-D ) and ( F, G ) except in separate cultures and % live cells (PI-ve) was assessed on days 4 and 6 (two-way ANOVA). Symbols in bar charts represent biological replicates, symbols in ( D ) represent the mean. Error bars show mean ± S.D. Data are representative of ( B, G ) n=4, ( C, F ) n=6 or show pooled data from ( D ) n=4, ( E ) n=6 biological replicates with data collected over at least two independent experiments. Figure 3—source data 1. Raw values plotted in .

Article Snippet: Antibody , anti-phospho STAT5 Y694 (Rabbit, monoclonal, C11C5) , Cell Signaling Technology , Cat # 9359 S, RRID: AB_823649 , intracellular stain 1:200; western blot 1:1000.

Techniques: Quantitative Proteomics, Cell Culture, Negative Control, Expressing, Activation Assay

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: ( A ) OT1 lymph node cell suspensions were SIINFEKL peptide activated for 36 hr, washed then cultured with no cytokine, IL-15 (20 ng/mL) or IL-2 (20 ng/mL) for 4 or 24 hr. Western blots of PIM1 (two isoforms of 44 and 34 kDa, non-specific band indicated by *), PIM2 (three isoforms of 40, 37, and 34 kDa) or pSTAT5 Y694 expression. ( B ) Schematic of cytokine driven memory and effector CD8 T cell expansion and differentiation cultures. Lymph node or spleen cell suspensions were activated for 2 days with TCR stimulus + cytokine, washed, then split daily into fresh media + cytokine. ( C ) WT (Ly5.1) and Pim dKO LN suspensions were mixed at a 50:50 ratio for T cells and cultured as outlined in ( B ) with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), and CD8 T cell number was measured daily. ( D ) WT and Pim dKO T cells were expanded with IL-15 in separate cultures as per ( B, C ) and % live cells (PI-ve) were assessed on days 4 and 6 (two-way ANOVA). ( E–J ) WT and Pim dKO CD8 T cells were activated with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), expanded with IL-15 as per ( B ), with an additional CD4 T cell magnetic depletion step on day 3 of culture. CD8 T cells were harvested on day 6 for parallel RNAseq and proteomic analysis. An interactive version of the proteomics expression data is available for exploration on the Immunological Proteome Resource website: immpres.co.uk ( E ) Fold-change in mRNA expression between Pim dKO and WT versus average mRNA expression (TPM). mRNA expression (Transcripts per million, TPM) of ( F ) secondary lymphoid homing receptors Sell, Ccr7, S1pr1 and ( G ) key transcription factors involved in CD8 T cell memory differentiation and maintenance Tcf7, Klf2, Foxo1, Foxo3, Id3. ( H ) WT vs Pim dKO protein copy numbers, differentially expression proteins (FC >1.5, q<0.05) are highlighted in red ( I ) Protein copy numbers per cell for key mitochondrial proteins DRP1, OPA1, and CPT1A. ( J ) WT vs Pim dKO protein copy numbers, mitochondrial proteins (as defined in MitoCarta 3.0) are highlight in pink. Symbols in bar charts represent biological replicates, symbols in ( C, E, H, J ) represent the mean. Error bars show mean ± S.D. Data are representative of ( A ) n=3 or show pooled data from ( C ) n=4, and ( D ) n=5 biological replicates with data collected over at least two independent experiments. Quantitative proteomics and RNAseq was performed on biological triplicates. ** q≤0.01, fold-change (FC) shown on bar graphs when q<0.05. Figure 2—source data 1. PDF files containing labelled and uncropped images for western blots displayed in . Figure 2—source data 2. Original files for western blot images displayed in . Figure 2—source data 3. Raw values plotted in .

Article Snippet: Blots were probed with the following primary antibodies: S6K (5G10, Cell Signaling Technology, cat# 2217 S), S6K p-T389 (108D2, Cell Signaling Technology, cat# 9234 S), STAT5 p-Y694 (C11C5, Cell Signalling Technology, cat# 9359 S), Pim1 (12H8, Santa Cruz, cat# SC-13513), Pim2 (1D12, Santa Cruz, cat# SC-13514).

Techniques: Cell Culture, Western Blot, Expressing, Quantitative Proteomics

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: ( A ) Estimated copies per cell of PIM1 and PIM2 protein from published quantitative proteomics analysis ; of CD8 T cells expanded in IL-2 or IL-15 as outlined in . ( B–D, F, G ) WT (Ly5.1) and Pim dKO lymph node or spleen single-cell suspensions were mixed at a 50:50 ratio of T cells, activated for 2 days with αCD3/αCD28 (both 0.5 µg/mL) and IL-2 (20 ng/mL), washed then split into fresh medium containing IL-2 (20 ng/mL) daily (as per ). Some of the mixed cell suspensions were also cultured in IL-7 (5 ng/mL) to sustain a naive T cell reference. ( B ) WT and Pim dKO CTL were treated 1 hr +/- Jak1/3 inhibitor Tofacitinib (100 nM; negative control) before pSTAT5 Y694 expression was measured on day 3 and 6 of culture, ( C ) surface CD25 expression was measured on days 3 and 6 of culture, ( D ) CD8 T cell number vs time was calculated, ( F ) CD8 T cell FSC-A, SSC-A and surface activation markers (CD44, CD71) were measured on days 3 and 6 of culture ( G ) expression of adhesion molecule CD62L was measured daily. ( E ) WT and Pim dKO T cells were activated and expanded with IL-2 as per ( B-D ) and ( F, G ) except in separate cultures and % live cells (PI-ve) was assessed on days 4 and 6 (two-way ANOVA). Symbols in bar charts represent biological replicates, symbols in ( D ) represent the mean. Error bars show mean ± S.D. Data are representative of ( B, G ) n=4, ( C, F ) n=6 or show pooled data from ( D ) n=4, ( E ) n=6 biological replicates with data collected over at least two independent experiments. Figure 3—source data 1. Raw values plotted in .

Article Snippet: Blots were probed with the following primary antibodies: S6K (5G10, Cell Signaling Technology, cat# 2217 S), S6K p-T389 (108D2, Cell Signaling Technology, cat# 9234 S), STAT5 p-Y694 (C11C5, Cell Signalling Technology, cat# 9359 S), Pim1 (12H8, Santa Cruz, cat# SC-13513), Pim2 (1D12, Santa Cruz, cat# SC-13514).

Techniques: Quantitative Proteomics, Cell Culture, Negative Control, Expressing, Activation Assay