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Cell Signaling Technology Inc pshp 1
Pshp 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pshp+1/pmc12871476-305-48-52?v=Cell+Signaling+Technology+Inc
Average 94 stars, based on 86 article reviews

pshp 1 - by Bioz Stars, 2026-07

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( A ) YTS-2DL1 NK cells were incubated with either inhibitory 721-Cw4 HLA or activating 721-Cw7 HLA target cells at 37°C for 5 min, and then lysed. Lysates were separated on SDS-PAGE and immunoblotted with anti-pSHP-1 S591 antibody. SHP-1 S591 phosphorylation levels were measured by densitometric analysis, relative to β-tubulin loading control using ImageJ. Samples were normalized to the YTS-2DL1 sample incubated with 721-Cw4 target after 5 min of activation (p=0.0104, quantification on the right showing the average of three independent experiments). ( B ) pNK-2DL1 cells were incubated with either 721-Cw4 HLA or 721-HLA-negative target cells at 37°C for 5 min. pSHP-1 S591 levels were determined as in ( A ) (p=0.0060, quantification on the right showing the average of three independent experiments). ( C ) YTS-2DL1 cells were incubated with target cells as described in ( A ), for four different time points, as indicated. pSHP-1 S591 levels were quantitated as in ( A ). Statistical significance between Cw4 and Cw7 after 5 min of activation (p=0.015), statistical significance between Cw4 at 5 min versus 20 min (p=0.0091). pSHP-1 S591 levels of YTS-2DL1 cells incubated with targets for 5 and 20 min are shown in the bar graph (quantification showing the average of three independent experiments). Data are shown as mean ± SEM. One-sample t -tests ( A, B ) or one-way ANOVA with Tukey test ( C ) was used to calculate p-values. Figure 1—source data 1. Representative blots. Figure 1—source data 2. Numerical data for all the graphical presentations in .

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/eLife.73282

Figure Lengend Snippet: ( A ) YTS-2DL1 NK cells were incubated with either inhibitory 721-Cw4 HLA or activating 721-Cw7 HLA target cells at 37°C for 5 min, and then lysed. Lysates were separated on SDS-PAGE and immunoblotted with anti-pSHP-1 S591 antibody. SHP-1 S591 phosphorylation levels were measured by densitometric analysis, relative to β-tubulin loading control using ImageJ. Samples were normalized to the YTS-2DL1 sample incubated with 721-Cw4 target after 5 min of activation (p=0.0104, quantification on the right showing the average of three independent experiments). ( B ) pNK-2DL1 cells were incubated with either 721-Cw4 HLA or 721-HLA-negative target cells at 37°C for 5 min. pSHP-1 S591 levels were determined as in ( A ) (p=0.0060, quantification on the right showing the average of three independent experiments). ( C ) YTS-2DL1 cells were incubated with target cells as described in ( A ), for four different time points, as indicated. pSHP-1 S591 levels were quantitated as in ( A ). Statistical significance between Cw4 and Cw7 after 5 min of activation (p=0.015), statistical significance between Cw4 at 5 min versus 20 min (p=0.0091). pSHP-1 S591 levels of YTS-2DL1 cells incubated with targets for 5 and 20 min are shown in the bar graph (quantification showing the average of three independent experiments). Data are shown as mean ± SEM. One-sample t -tests ( A, B ) or one-way ANOVA with Tukey test ( C ) was used to calculate p-values. Figure 1—source data 1. Representative blots. Figure 1—source data 2. Numerical data for all the graphical presentations in .

Article Snippet: Antibody , Anti-human pSHP-1 (S591) (rabbit polyclonal) , ECM Biosciences , Sp-1531 , IB: 1:1000 (7 μL)IF: 1:500 (0.6 μL).

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

( A ) pNK-2DL1 cells were incubated with target cells as described in , for two different activation time points, as indicated. pSHP-1 S591 levels were determined as in . pSHP-1 S591 levels of pNK-2DL1 cells incubated with targets for 5 and 20 min are shown on the bar graph. The bar graph shows the average of four independent experiments. Figure 1—figure supplement 1—source data 1. Numerical data for the graphical presentation in .

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/eLife.73282

Figure Lengend Snippet: ( A ) pNK-2DL1 cells were incubated with target cells as described in , for two different activation time points, as indicated. pSHP-1 S591 levels were determined as in . pSHP-1 S591 levels of pNK-2DL1 cells incubated with targets for 5 and 20 min are shown on the bar graph. The bar graph shows the average of four independent experiments. Figure 1—figure supplement 1—source data 1. Numerical data for the graphical presentation in .

Article Snippet: Antibody , Anti-human pSHP-1 (S591) (rabbit polyclonal) , ECM Biosciences , Sp-1531 , IB: 1:1000 (7 μL)IF: 1:500 (0.6 μL).

Techniques: Incubation, Activation Assay

( A ) pNK-2DL1 cells were incubated over slides pre-seeded with 721-Cw4 and K562 cells expressing mCherry or CFP, respectively. The cells were incubated for 5 min at 37°C to enable conjugate formation and were fixed. pSHP-1 S591 was labeled with primary rabbit anti-pSHP-1 S591 antibody, and secondary anti-rabbit 488 antibody. Synapse intensity was quantified in NK cells relative to each cell in multiple NK cell synapses with two different targets (p=0.007, quantification on the right of triple-cell conjugates collected, n = 26). ( B ) YTS-2DL1 cells were incubated over slides pre-seeded with 721-Cw4 target cells expressing mCherry. The cells were incubated for 5 or 20 min at 37°C to enable conjugate formation and were fixed. PKC-θ was subsequently labeled with primary goat anti-PKC-θ antibody and secondary anti-goat 488 antibody. Right: graph summarizing PKC-θ accumulation at the NKIS at two time points following activation. Analysis was conducted comparing PKC-θ intensity at the NKIS relative to the rest of the NK cell. For Cw4, 5 and 20 min activation, n = 24 cell conjugates were analyzed from three independent experiments. Data are shown as mean ± SEM. One-sample t -tests ( A, B ) were used to calculate p-values. Figure 3—source data 1. Numerical data for all graphical presentations in .

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/eLife.73282

Figure Lengend Snippet: ( A ) pNK-2DL1 cells were incubated over slides pre-seeded with 721-Cw4 and K562 cells expressing mCherry or CFP, respectively. The cells were incubated for 5 min at 37°C to enable conjugate formation and were fixed. pSHP-1 S591 was labeled with primary rabbit anti-pSHP-1 S591 antibody, and secondary anti-rabbit 488 antibody. Synapse intensity was quantified in NK cells relative to each cell in multiple NK cell synapses with two different targets (p=0.007, quantification on the right of triple-cell conjugates collected, n = 26). ( B ) YTS-2DL1 cells were incubated over slides pre-seeded with 721-Cw4 target cells expressing mCherry. The cells were incubated for 5 or 20 min at 37°C to enable conjugate formation and were fixed. PKC-θ was subsequently labeled with primary goat anti-PKC-θ antibody and secondary anti-goat 488 antibody. Right: graph summarizing PKC-θ accumulation at the NKIS at two time points following activation. Analysis was conducted comparing PKC-θ intensity at the NKIS relative to the rest of the NK cell. For Cw4, 5 and 20 min activation, n = 24 cell conjugates were analyzed from three independent experiments. Data are shown as mean ± SEM. One-sample t -tests ( A, B ) were used to calculate p-values. Figure 3—source data 1. Numerical data for all graphical presentations in .

Article Snippet: Antibody , Anti-human pSHP-1 (S591) (rabbit polyclonal) , ECM Biosciences , Sp-1531 , IB: 1:1000 (7 μL)IF: 1:500 (0.6 μL).

Techniques: Incubation, Expressing, Labeling, Activation Assay

( A ) Silencing efficiency of PKC-θ. YTS-2DL1 cells treated with either nonspecific (control) (NS) or PKC-θ siRNA, lysed, separated on SDS-PAGE, and immunoblotted with anti- PKC-θ antibody. PKC-θ levels were measured by densitometric analysis, relative to the GAPDH loading control using ImageJ. Samples were normalized to the sample treated with NS siRNA (p=0.001, quantification on the bottom showing average of three independent experiments). ( B ) YTS-2DL1 treated with either NS or PKC-θ siRNA were incubated with 721- HLA target cells at 37°C for 5 min, and cells were subsequently lysed. Lysates were separated on SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with anti-pSHP-1 S591 antibody. SHP-1 S591 phosphorylation levels were measured by densitometric analysis, relative to the GAPDH loading control using ImageJ. Samples were normalized to the YTS-2DL1 sample treated with NS siRNA and incubated with 721-Cw7 targets (p=0.0072, quantification on the bottom of independent experiments, n = 3). ( C ) Silencing efficiency of PKC-θ. pNK-2DL1 cells were transfected with 250 pmol of PKC-θ siRNA. After 48 hr, prior to incubation with target cells, pNK-2DL1 were counted and lysed. Lysates were separated on SDS-PAGE and immunoblotted with anti-PKC-θ antibody. PKC-θ expression levels were measured by densitometric analysis using ImageJ and expressed relative to the GAPDH loading control. Samples were normalized according to the pNK-NS siRNA sample. Bar graph on the bottom shows the average of three independent experiments. ( D ) pNK-2DL1 cells were incubated with 721-HLA-negative cells at 37°C for 5 min, and cells were subsequently lysed. Lysates were separated on SDS-PAGE and transferred to a nitrocellulose membrane that was immunoblotted with anti-pSHP-1 S591 antibody. SHP-1 S591 phosphorylation levels were measured by densitometric analysis, relative to the GAPDH loading control using ImageJ. Samples were normalized to the pNK-2DL1 sample treated with NS siRNA and incubated with 721 targets (p=0.0113, quantification on the bottom showing the average of three independent experiments). Data are shown as mean ± SEM. One-sample t -tests ( A–D ) were used to calculate p-values. Figure 4—source data 1. Representative blots. Figure 4—source data 2. Numerical data for all the graphical presentations in .

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/eLife.73282

Figure Lengend Snippet: ( A ) Silencing efficiency of PKC-θ. YTS-2DL1 cells treated with either nonspecific (control) (NS) or PKC-θ siRNA, lysed, separated on SDS-PAGE, and immunoblotted with anti- PKC-θ antibody. PKC-θ levels were measured by densitometric analysis, relative to the GAPDH loading control using ImageJ. Samples were normalized to the sample treated with NS siRNA (p=0.001, quantification on the bottom showing average of three independent experiments). ( B ) YTS-2DL1 treated with either NS or PKC-θ siRNA were incubated with 721- HLA target cells at 37°C for 5 min, and cells were subsequently lysed. Lysates were separated on SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with anti-pSHP-1 S591 antibody. SHP-1 S591 phosphorylation levels were measured by densitometric analysis, relative to the GAPDH loading control using ImageJ. Samples were normalized to the YTS-2DL1 sample treated with NS siRNA and incubated with 721-Cw7 targets (p=0.0072, quantification on the bottom of independent experiments, n = 3). ( C ) Silencing efficiency of PKC-θ. pNK-2DL1 cells were transfected with 250 pmol of PKC-θ siRNA. After 48 hr, prior to incubation with target cells, pNK-2DL1 were counted and lysed. Lysates were separated on SDS-PAGE and immunoblotted with anti-PKC-θ antibody. PKC-θ expression levels were measured by densitometric analysis using ImageJ and expressed relative to the GAPDH loading control. Samples were normalized according to the pNK-NS siRNA sample. Bar graph on the bottom shows the average of three independent experiments. ( D ) pNK-2DL1 cells were incubated with 721-HLA-negative cells at 37°C for 5 min, and cells were subsequently lysed. Lysates were separated on SDS-PAGE and transferred to a nitrocellulose membrane that was immunoblotted with anti-pSHP-1 S591 antibody. SHP-1 S591 phosphorylation levels were measured by densitometric analysis, relative to the GAPDH loading control using ImageJ. Samples were normalized to the pNK-2DL1 sample treated with NS siRNA and incubated with 721 targets (p=0.0113, quantification on the bottom showing the average of three independent experiments). Data are shown as mean ± SEM. One-sample t -tests ( A–D ) were used to calculate p-values. Figure 4—source data 1. Representative blots. Figure 4—source data 2. Numerical data for all the graphical presentations in .

Article Snippet: Antibody , Anti-human pSHP-1 (S591) (rabbit polyclonal) , ECM Biosciences , Sp-1531 , IB: 1:1000 (7 μL)IF: 1:500 (0.6 μL).

Techniques: Control, SDS Page, Incubation, Membrane, Phospho-proteomics, Transfection, Expressing

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/eLife.73282

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human pSHP-1 (S591) (rabbit polyclonal) , ECM Biosciences , Sp-1531 , IB: 1:1000 (7 μL)IF: 1:500 (0.6 μL).

Techniques: Recombinant, Plasmid Preparation, CRISPR, Sequencing, Selection, Software, In Vivo

Figure 1. Phosphorylation kinetics of SHP-1 S591 during activating and inhibitory NK cell interactions. (A) YTS-2DL1 NK cells were incubated with either inhibitory 721-Cw4 HLA or activating 721-Cw7 HLA target cells at 37°C for 5 min, and then lysed. Lysates were separated on SDS-PAGE and immunoblotted with anti-pSHP-1 S591 antibody. SHP-1 S591 phosphorylation levels were measured by densitometric analysis, relative to β-tubulin loading control using ImageJ. Samples were normalized to the YTS-2DL1 sample incubated with 721-Cw4 target after 5 min of activation (p=0.0104, quantification on the right showing the average of three independent experiments). (B) pNK-2DL1 cells were incubated with either 721-Cw4 HLA or 721-HLA-negative target cells at 37°C for 5 min. pSHP-1 S591 levels were determined as in (A) (p=0.0060, quantification on the right showing the average of three independent experiments). (C) YTS-2DL1 cells were incubated with target cells as described in (A), for four different time points, as indicated. pSHP-1 S591 levels were quantitated as in (A). Statistical significance between Cw4 and Cw7 after 5 min of activation (p=0.015), statistical significance between Cw4 at 5 min versus 20 min (p=0.0091). pSHP-1 S591 levels of YTS-2DL1 cells incubated with targets for 5 and 20 min are shown in the bar graph (quantification showing the average of three independent experiments). Data are shown as mean ± SEM. One-sample t-tests (A, B) or one-way ANOVA with Tukey test (C) was used to calculate p-values.

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/elife.73282

Figure Lengend Snippet: Figure 1. Phosphorylation kinetics of SHP-1 S591 during activating and inhibitory NK cell interactions. (A) YTS-2DL1 NK cells were incubated with either inhibitory 721-Cw4 HLA or activating 721-Cw7 HLA target cells at 37°C for 5 min, and then lysed. Lysates were separated on SDS-PAGE and immunoblotted with anti-pSHP-1 S591 antibody. SHP-1 S591 phosphorylation levels were measured by densitometric analysis, relative to β-tubulin loading control using ImageJ. Samples were normalized to the YTS-2DL1 sample incubated with 721-Cw4 target after 5 min of activation (p=0.0104, quantification on the right showing the average of three independent experiments). (B) pNK-2DL1 cells were incubated with either 721-Cw4 HLA or 721-HLA-negative target cells at 37°C for 5 min. pSHP-1 S591 levels were determined as in (A) (p=0.0060, quantification on the right showing the average of three independent experiments). (C) YTS-2DL1 cells were incubated with target cells as described in (A), for four different time points, as indicated. pSHP-1 S591 levels were quantitated as in (A). Statistical significance between Cw4 and Cw7 after 5 min of activation (p=0.015), statistical significance between Cw4 at 5 min versus 20 min (p=0.0091). pSHP-1 S591 levels of YTS-2DL1 cells incubated with targets for 5 and 20 min are shown in the bar graph (quantification showing the average of three independent experiments). Data are shown as mean ± SEM. One-sample t-tests (A, B) or one-way ANOVA with Tukey test (C) was used to calculate p-values.

Article Snippet: DOI: https://doi.org/10.7554/eLife.73282 19 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- human VAV1 (D7) (mouse monoclonal) Santa Cruz SC- 8039 IB: 1:500 (14 μL) Antibody Anti- human SHP- 1 (SH- PTP- 1) (C- 19) (rabbit polyclonal) Santa Cruz SC- 287 IB: 1:1000 (7 μL) Antibody Anti- human GAPDH (0411) (mouse monoclonal) Santa Cruz SC- 47724 IB: 1:1000 (7 μL) Antibody Anti- human pSHP- 1 (S591) (rabbit polyclonal) ECM Biosciences Sp- 1531 IB: 1:1000 (7 μL) IF: 1:500 (0.6 μL) Antibody Anti- human pPLCγ (Y783) (rabbit polyclonal) Cell Signaling CST- 2821S IB: 1:1000 (7 μL) IF: 1:500 (0.6 μL) Antibody Anti- human pVAV1 (Y160) (rabbit polyclonal) Bio Source Bs- 44482 IB: 1:1000 (7 μL) IF: 1:500 (0.6 μL) Antibody Anti- human- PKC-θ (1C2) (mouse monoclonal) Santa Cruz SC- 81534 IB: 1:500 (14 μL) IF: 1:250 (1.2 μL) Antibody Goat anti- mouse Jackson Laboratory #115- 035- 003 1:10,000 (1 μL) Antibody Goat anti- rabbit Santa Cruz Sc- 2004 1:10,000 (1 μL) Antibody Anti- human KIR2DL1/ S1- PE conjugated (mouse monoclonal) Miltenyi Biotec 130- 099- 209 1:10 (10 μL) Antibody Anti- human CD107a (LAMP- 1) (mouse monoclonal) BioLegend #328602 1:20 (2.5 μL) Antibody Alexa Fluor- conjugated 488 (goat polyclonal) anti- rabbit IgG (H+L) Highly CrossAdsorbed Secondary Antibody Invitrogen A11034 IF: 1:2000 (1 μL) Antibody Alexa Fluor- conjugated 488 goat polyclonal anti- mouse IgG (H+L) Jackson Laboratory 115- 545- 146 IF: 1:2000 (1 μL) Recombinant DNA reagent YFP- SHP- 1- CFP (plasmid) Matalon et al., 2018 Recombinant DNA reagent CRISPR CAS9 SHP- 1 S591D Ben- Shmuel et al., 2021 Backbone pSpCas9 (BB)–2A- GFP vector Addgene plasmid #48138 Sequence- based reagent siRNA: target PRKCQ gene Sigma- Aldrich 5′ CUCUUCACCUGGGCGCCAA 3′ 5′ UUGGCGCCCAGGUGAAGAG 3′ Sequence- based reagent siRNA: nonspecific target Sigma- Aldrich 5′ UAGCGACUAAACACAUCAA 3′, 5′UAAGGCUAUGAAGAGAUAC3′, 5′AUGUAUUGGCCUGUAUUAG3′, 5′ AUGAACGUGAAUUGCUCAA 3′, and 5′ UGGUUUACAUGUCGACUAA3′ Chemical compound, drug Fluo- 3- AM Biotium 50016 1 μg per sample Chemical compound, drug p- Nitophenyl phosphate (pNPP) NEB- P0757S Chemical compound, drug [35S]Met PerkinElmer NEG009L005MC Chemical compound, drug Monensin BioLegend #420701 Chemical reagent Mirus Ingenio Solution MIR50111 Chemical compound, reagent Enhanced chemiluminescence PerkinElmer, Life Gene NEL105001EA, AC2103 Continued on next page Ben- Shmuel, Sabag, et al. eLife 2022;11:e73282.

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

Figure 2. SHP-1 conformational dynamics reflect S591 phosphorylation during activating and inhibitory NK cell interactions. (A) YTS-2DL1 YFP-SHP-1-CFP cells were incubated over slides pre-seeded with 721-Cw4 (top panels) or Cw7 (bottom panels) target cells expressing mCherry. The cells were incubated for 5 or 20 min at 37°C to enable conjugate formation, and fixed. FRET analysis was performed as indicated. (B) Graph summarizing FRET efficiency following 5 or 20 min activation with Cw4 or Cw7 target cells. For Cw4, 5 and 20 min activation, n = 72 and 62 cell conjugates analyzed, respectively. For Cw7, 5 and 20 min activation, n = 73 and 47 cell conjugates analyzed from three independent experiments, respectively. Data are shown as mean ± SEM. Two-way ANOVA with Tukey test (B) was used to calculate p-values.

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/elife.73282

Figure Lengend Snippet: Figure 2. SHP-1 conformational dynamics reflect S591 phosphorylation during activating and inhibitory NK cell interactions. (A) YTS-2DL1 YFP-SHP-1-CFP cells were incubated over slides pre-seeded with 721-Cw4 (top panels) or Cw7 (bottom panels) target cells expressing mCherry. The cells were incubated for 5 or 20 min at 37°C to enable conjugate formation, and fixed. FRET analysis was performed as indicated. (B) Graph summarizing FRET efficiency following 5 or 20 min activation with Cw4 or Cw7 target cells. For Cw4, 5 and 20 min activation, n = 72 and 62 cell conjugates analyzed, respectively. For Cw7, 5 and 20 min activation, n = 73 and 47 cell conjugates analyzed from three independent experiments, respectively. Data are shown as mean ± SEM. Two-way ANOVA with Tukey test (B) was used to calculate p-values.

Techniques: Phospho-proteomics, Incubation, Expressing, Activation Assay

Figure 4. SHP-1 phosphorylation is mediated through PKC-θ. (A) Silencing efficiency of PKC-θ. YTS-2DL1 cells treated with either nonspecific (control) (NS) or PKC-θ siRNA, lysed, separated on SDS-PAGE, and immunoblotted with anti- PKC-θ antibody. PKC-θ levels were measured by densitometric analysis, relative to the GAPDH loading control using ImageJ. Samples were normalized to the sample treated with NS siRNA (p=0.001, quantification on the bottom showing average of three independent experiments). (B) YTS-2DL1 treated with either NS or PKC-θ siRNA were incubated with 721- HLA target cells at 37°C for 5 min, and cells were subsequently lysed. Lysates were separated on SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with anti-pSHP-1 S591 antibody. SHP-1 S591 phosphorylation levels were measured by densitometric analysis, relative to the GAPDH loading control using ImageJ. Samples were normalized to the YTS-2DL1 sample treated with NS siRNA and incubated with 721-Cw7 targets (p=0.0072, quantification on the bottom of independent experiments, n = 3). (C) Silencing efficiency of PKC-θ. pNK-2DL1 cells were transfected with 250 pmol of PKC-θ siRNA. After 48 hr, prior to incubation with target cells, pNK-2DL1 were counted and lysed. Lysates were separated on SDS-PAGE and immunoblotted with anti-PKC-θ antibody. PKC-θ expression levels were measured by densitometric analysis using ImageJ and expressed relative to the GAPDH loading control. Samples were normalized according to the pNK-NS siRNA sample. Bar graph on the bottom shows the average of three independent experiments. (D) pNK-2DL1 cells were incubated with 721-HLA-negative cells at 37°C for 5 min, and cells were subsequently lysed. Lysates were separated on SDS-PAGE and transferred to a nitrocellulose membrane that was immunoblotted with anti-pSHP-1 S591 antibody. SHP-1 S591 phosphorylation levels were measured by densitometric analysis, relative to the GAPDH loading control using ImageJ. Samples were normalized to the pNK-2DL1 sample treated with NS siRNA and incubated with 721 targets (p=0.0113, quantification on the bottom showing the average of three independent experiments). Data are shown as mean ± SEM. One-sample t-tests (A–D) were used to calculate p-values.

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/elife.73282

Figure Lengend Snippet: Figure 4. SHP-1 phosphorylation is mediated through PKC-θ. (A) Silencing efficiency of PKC-θ. YTS-2DL1 cells treated with either nonspecific (control) (NS) or PKC-θ siRNA, lysed, separated on SDS-PAGE, and immunoblotted with anti- PKC-θ antibody. PKC-θ levels were measured by densitometric analysis, relative to the GAPDH loading control using ImageJ. Samples were normalized to the sample treated with NS siRNA (p=0.001, quantification on the bottom showing average of three independent experiments). (B) YTS-2DL1 treated with either NS or PKC-θ siRNA were incubated with 721- HLA target cells at 37°C for 5 min, and cells were subsequently lysed. Lysates were separated on SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with anti-pSHP-1 S591 antibody. SHP-1 S591 phosphorylation levels were measured by densitometric analysis, relative to the GAPDH loading control using ImageJ. Samples were normalized to the YTS-2DL1 sample treated with NS siRNA and incubated with 721-Cw7 targets (p=0.0072, quantification on the bottom of independent experiments, n = 3). (C) Silencing efficiency of PKC-θ. pNK-2DL1 cells were transfected with 250 pmol of PKC-θ siRNA. After 48 hr, prior to incubation with target cells, pNK-2DL1 were counted and lysed. Lysates were separated on SDS-PAGE and immunoblotted with anti-PKC-θ antibody. PKC-θ expression levels were measured by densitometric analysis using ImageJ and expressed relative to the GAPDH loading control. Samples were normalized according to the pNK-NS siRNA sample. Bar graph on the bottom shows the average of three independent experiments. (D) pNK-2DL1 cells were incubated with 721-HLA-negative cells at 37°C for 5 min, and cells were subsequently lysed. Lysates were separated on SDS-PAGE and transferred to a nitrocellulose membrane that was immunoblotted with anti-pSHP-1 S591 antibody. SHP-1 S591 phosphorylation levels were measured by densitometric analysis, relative to the GAPDH loading control using ImageJ. Samples were normalized to the pNK-2DL1 sample treated with NS siRNA and incubated with 721 targets (p=0.0113, quantification on the bottom showing the average of three independent experiments). Data are shown as mean ± SEM. One-sample t-tests (A–D) were used to calculate p-values.

Techniques: Phospho-proteomics, Control, SDS Page, Incubation, Membrane, Transfection, Expressing

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/eLife.73282

Article Snippet: Antibodies and their sources were as follows: mouse anti-PLCγ1 (Upstate), mouse anti-VAV1 (D7), rabbit anti-SHP-1 and mouse anti-GAPDH (Santa Cruz), rabbit anti-pSHP-1 (S591) (ECM Biosciences), rabbit anti-pPLCγ1Y783 (Bio Source), rabbit anti-pVAV-1Y160 (Bio Source), and mouse anti-PKC-θ (Santa Cruz).

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

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/eLife.73282

Techniques: Incubation, Activation Assay

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/eLife.73282

Techniques: Incubation, Expressing, Labeling, Activation Assay

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/eLife.73282

Techniques: Control, SDS Page, Incubation, Membrane, Phospho-proteomics, Transfection, Expressing

Journal: eLife

Article Title: Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

doi: 10.7554/eLife.73282

Figure Lengend Snippet:

Techniques: Recombinant, Plasmid Preparation, CRISPR, Sequencing, Selection, Software, In Vivo

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