pshp 1  (Cell Signaling Technology Inc)

Journal: The EMBO Journal

Article Title: Dysfunctional natural killer cells can be reprogrammed to regain anti-tumor activity

doi: 10.1038/s44318-024-00094-5

Figure Lengend Snippet: ( A ) Gene silencing of DGKα. Anergic cells were treated with DGKα siRNA or NS siRNA and were then lysed and subjected to western blot analysis. One blot representative of three experiments is shown. The right panel shows a graph representing the quantified blots ( n = 3, where n is the number of healthy donors used to obtain the pNK cells). Data are presented as mean ± SEM . P value was calculated using a two-tailed paired t test and is indicated within the graph. ( B ) Gating strategy employed for the analysis of results in Fig. . The NK cells and the 221 HLA-Cw7 cells were differentiated based on size and granularity (FSC-SSC) and gated for PE to distinguish the anergic (PE − ) versus the responsive population (PE + ). They were subsequently gated for CD107a, as indicated on the overlaid histograms. ( C – E ) Purified responsive and anergic NK cells were stimulated with 721.221 target cells, lysed and subjected to FACS analysis with ( C ) anti-pSHP-1(S591), ( D ) anti-pPLCγ1 (Y783), and ( E ) anti-pPLCγ2 (Y1217) antibodies. Graph summarizing the MFI of pSHP-1 ( n = 5), pPLCγ1 ( n = 3), and pPLCγ2 ( n = 5) expression levels (where n is the number of healthy donors used to obtain the pNK cells). Data are presented as mean ± SEM. P value was calculated using a two-tailed t test with matched data repeats and is indicated within the graph. ( F ) Representative overlaid histograms for Fig. . .

Article Snippet: Primary antibodies for immunoprecipitation and immunoblotting: rabbit anti-Cbl, mouse anti-GAPDH (sc-0411), rabbit anti-GAPDH (FL-335), mouse anti-pERK Tyr204 (Santa Cruz Biotechnology); rabbit anti-Egr2 (Sigma Aldrich), rabbit anti-DGKα (Proteintech); mouse anti-NFAT1 (Abcam), mouse anti- NFAT2 (Abcam), rabbit anti-DGKζ (Abcam); rabbit anti-pPLCγ1(Y783), rabbit anti-pSHP-1 S591(ECM Biosciences).

Techniques: Western Blot, Two Tailed Test, Purification, Expressing

Journal: The EMBO Journal

Article Title: Dysfunctional natural killer cells can be reprogrammed to regain anti-tumor activity

doi: 10.1038/s44318-024-00094-5

Figure Lengend Snippet: ( A , B ) Correlation of NK- DGKA-EGR2 signatures with the overall survival of ( A ) AML patients, and ( B ) glioma patients comparing high and low quartiles. Kaplan Meier curves are presented showing patient survival (obtained from Survival Genie (Dwivedi et al, )) along with the P values. Right panels show the signature of immune cell infiltration in AML and glioma; and red squares represent positive correlation to immune cells observed in the cancer dataset. Bottom panels indicate the correlation of the survival score to the NK cell signatures. P values ( A , B ) were calculated using Log-rank t test. ( C ) Left panel: Schematic representation of the 3D OTS model using the NP delivery platform to reprogram anergic NK cells in a tumor milieu. Primary NK cells were used to obtain anergic and responsive subsets and were then seeded to pre-constructed 3D cultures and administered with NP encapsulating Egr2 or NS siRNA. Next, the pNKs were subjected to an Incucyte-based killing assay. Right panel: Human chronic myeloid leukemia OTS 3D domes were established using Matrigel and cultured for 48 h in OTS media consisting of RPMI media supplemented with 1 µg/mL fibroblast growth factor (FGF), 0.18 µg/mL epidermal growth factor (EGF) and 500 IU/mL transforming growth factor (TGFβ). After 48 h, the 3D cultures were incubated with either freshly isolated anergic or responsive NK cells. After 6 h of NK-tumor co-incubation, NPs encapsulating Egr2 siRNA or NS siRNA were added (represented by the pink-shaded region). The decrease in fluorescence intensity reflects target cell lysis by the respective NK cell population and the associated treatment. The analysis was performed for 25 individual field frames for each experimental condition (with approx. n = 30 cells each field). Statistical analysis was conducted for three independent experiments, and presented as means ± SEM ( n = 3 healthy donors). P value was calculated using two-way ANOVA with a Tukey’s post hoc test for multiple comparison, and paired t test was performed between each time point and group to find significant changes in tumor lysis between the groups at specific time points. Anergic NS siRNA vs Anergic Egr2 siRNA ( P value (*) = 0.0121, time point: 30 h). ( D ) Timeline of the in vivo experiment. PDAC-1-xenograft NRG mice were established as previously described and received a single infusion of 1.1*10 7 human pNK from four healthy donors on day 9 once the tumors reached ~250–300 cm 2 ; “tumor only” control did not receive NK cells but received NS siRNA encapsulated NPs. NPs encapsulating Egr2 siRNA or NS siRNA were administered i.v. from day 12 for every 3 days until day 27. ( E ) Tumor volume (in mm 3 ) was monitored and measured daily throughout the experiment. The pNK injection is depicted by a black arrow in a green-shaded region, and the NP injections are indicated by red arrows. Mice in the “tumor only” group ( n = 6 mice) received no pNK treatment but were administered NP encapsulated NS siRNA. Mice that were administered with Egr2 siRNA ( n = 8 mice) are depicted in red, while those receiving NS siRNA ( n = 8 mice) are depicted in blue. The bold lines indicate the average, and the dashed lines represent individual mice within their respective experimental groups. ( F ) Tumor sizes (mm 3 ) measured during the indicated days. Black graph represents the control group with tumor only. The blue and the red graphs represent groups of mice that received treatment with NP encapsulating NS siRNA or Egr2 siRNA, respectively. Tumor sizes at specific time points are indicated: Day 8 (one day before pNK injection) and Day 11 (2 days after pNK injection) are tumor sizes prior to NP injection; day 15 (6 days after pNK injection), day 19 (10 days after pNK injection) and day 27 (18 days after pNK injection and final day before tumor excision) represent tumor sizes following NP injection. Data are presented as mean ± SEM . P values are calculated using one-way ANOVA accompanied by a Tukeys’ post hoc multiple comparison test individually for each day presented and are indicated within the graph. ( G ) Tumor growth rate measured from day 12 (first NP administration) until day 27 (end point) shown for the three groups (Egr2 siRNA-NP vs NS siRNA-NP vs tumor only). Data are presented as mean ± SEM . P values are calculated using one-way ANOVA and are indicated within the graph ( N = 4 healthy donors were used to obtain the pNK; Groups – tumor only ( n = 6), NP Egr2 siRNA ( n = 8), NP NS siRNA ( n = 8), where n is the number of mice). ( H , I ) Ex vivo analysis. The tumors were excised on day 27 and processed to single-cell suspensions by dissociation as described in the Materials and Methods. They were then stained for ( H ) CD107a ( n = 3, where n is the number of mice used to obtain the pNK cells) and ( I ) PD-1 ( n = 3, where n is the number of mice used to obtain the pNK cells). The pNK were distinguished based on hCD45 expression and NP incorporation (PE positive). Fluorescence is represented as both relative MFI (left panels) and percentage of pNK (right panel). P values were calculated using a two-tailed paired t test and are represented within the graph presented as means ± SEM. ( J ) Scheme depicting the proposed signaling pathway of anergic cells in accordance with the transcriptome and protein level profiling; PA phosphatidic acid, PLC phospholipase Cγ1/2, DAG diacylglycerol, DGK diacylglycerol kinase, Egr early growth response, MAPK mitogen-activated protein kinase, NFAT nuclear factor of activated T cells, PD-1 programmed cell death protein 1, PKCθ protein kinase Cθ, SHP-1 Src homology 2 domain-containing protein tyrosine phosphatase 1, pS591 phospho – S591. “Anergic” cells exhibit elevated EGR2 expression, which subsequently triggers an increase in DGKα. This leads to the conversion of DAG to PA, in turn, PA recruits more SHP-1 to the cellular membrane. As a result availability of DAG is restricted, which hampers PKCθ activity, rendering it incapable of modulating SHP-1 activity (Ben-Shmuel et al, ). This allows SHP-1 to dephosphorylate LAT and PLCγ1/2 (Matalon et al, ), preventing the initiation of a secondary cascade. DAG depletion also inhibits the activation of the DAG-mediated Ras-Raf-MEK-ERK pathway and IP3-mediated calcium flux. Consequently, there is no nuclear translocation of NFAT and its effector partners, such as AP-1, to initiate an effector response. This ultimately results in establishing an “anergy-associated gene transcription program”. .

Article Snippet: Primary antibodies for immunoprecipitation and immunoblotting: rabbit anti-Cbl, mouse anti-GAPDH (sc-0411), rabbit anti-GAPDH (FL-335), mouse anti-pERK Tyr204 (Santa Cruz Biotechnology); rabbit anti-Egr2 (Sigma Aldrich), rabbit anti-DGKα (Proteintech); mouse anti-NFAT1 (Abcam), mouse anti- NFAT2 (Abcam), rabbit anti-DGKζ (Abcam); rabbit anti-pPLCγ1(Y783), rabbit anti-pSHP-1 S591(ECM Biosciences).

Techniques: Construct, Cell Culture, Incubation, Isolation, Fluorescence, Lysis, Comparison, In Vivo, Control, Injection, Ex Vivo, Staining, Expressing, Two Tailed Test, Membrane, Activity Assay, Activation Assay, Translocation 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

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

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

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

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

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

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.

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, Expressing, 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

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.

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, 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

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: 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

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: 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, 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

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: 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, 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

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