anti dap5 antibody  (Santa Cruz Biotechnology)

Journal: Endocrinology

Article Title: Robust serotonin activation of the kisspeptin GnRH pulse generator in male and female mice

doi: 10.1210/endocr/bqag034

Figure Lengend Snippet: Effects of serotonin and 5-HT receptor compounds on ARN KISS neuron firing in acute brain slices from diestrous female mice. (A) Robust excitatory effect of 90 seconds puff of 40 µM serotonin on firing rate of a middle ARN kisspeptin neuron. (B) Inhibitory effect of 90 seconds puff of 60 µM serotonin on firing rate of a caudal ARN kisspeptin neuron initially stimulated to fire by a 200 nM puff of NKB. (C) Rostral ARN kisspeptin neuron not responding to 60 µM puffs of serotonin but later activated by NKB. (D) Summary of percentage of rostral (rARN), middle (mARN), and caudal (cARN) kisspeptin neurons excited or inhibited by serotonin. (E) Caudal ARN kisspeptin neuron activated by serotonin (40 µM) in the absence and presence of methiothepin (100 µM). (F) Middle ARN kisspeptin neuron in which the excitatory effect of serotonin (40 µM) is blocked by methiothepin (100 µM). (G) Middle ARN kisspeptin neuron activated by serotonin (40 µM) in the absence and presence of SB228357 (100 µM). (H) Caudal ARN kisspeptin neuron in which zacopride (2 µM) facilitates serotonin excitation. (I) Whole-cell recording from an ARN KISS neuron in the continuous presence of TTX, CNQX, DAP5, and bicuculline, showing depolarization during bath application of 5-HT (30 µM). (J) Summary of mean membrane potential measured predrug, during 5-HT application, and following wash ( P < .001; n = 4 animals).

Article Snippet: Stock solutions of GABAzine (SR95531, 5 mM, Tocris, UK), CNQX (10 mM, Tocris, UK), and DAP5, (50 mM, Tocris, UK) were prepared with Milli-Q water or NaOH for DAP5.

Techniques: Membrane

Journal: Advanced Science

Article Title: Targeting DAP5 Disrupts Alternate Mode of Translational Initiation in Tregs and Potentiates Antitumor Immunity

doi: 10.1002/advs.202520625

Figure Lengend Snippet: Elevated DAP5 expression in ti‐Tregs is associated with ti‐Treg infiltrations. a) Western blotting results comparing activities of AKT/mTOR signaling pathway across naïve CD4 + T, iTh1, iTh2, iTh17 and iTregs. b) Flow cytometric results confirming reduced phosphorylation levels of Akt, S6K and 4E‐BP1 in mouse splenic Tregs than in conventional T cells (Tconv). c) Flow cytometric results revealing increased mean fluorescent intensities (MFI) of p‐eIF‐2α and Atf4 in ti‐Tregs than in pTregs. d) Box plots displaying higher puromycin‐labeling intensities among ti‐Tregs than in pTregs. e) Left panel: the representative histograms depicting distribution of Dap5 expressions among pTregs and ti‐Tregs; right panel: dot plots showing pairwise comparison of Dap5 MFI between ti‐Tregs and pTregs. f) The MFI of DAP5 among tiTregs compared with that in Tregs from paracancerous tissues. The p ‐value was determined by performing two‐tailed paired T test. g) Scatter plot displaying positive correlation between the DAP5 MFI and ti‐Treg frequencies in CRCs. p‐ values were determined by two‐tailed paired Student's T ‐test (b, c, e and f), unpaired Student's T ‐test (d) and Spearman's correlation Test (g), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: To dissect the function of Dap5 in Tregs in vivo, we generated transgenic mice with specific Dap5 ablation in Tregs ( Dap5 ΔFoxp3 ) by crossing the floxed‐ Dap5 mice with the Foxp3 ‐ Cre (B6.129(Cg)‐ Foxp3 4(YFP/icre)Ayr /J) strain from Jackson Laboratory that are widely used in Treg studies (Figure , Supporting Information).

Techniques: Expressing, Western Blot, Phospho-proteomics, Labeling, Comparison, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting DAP5 Disrupts Alternate Mode of Translational Initiation in Tregs and Potentiates Antitumor Immunity

doi: 10.1002/advs.202520625

Figure Lengend Snippet: Ablation of Dap5 in Tregs did not affect tTreg development and pTreg differentiation in vivo. a) Naïve CD4 + T cells were purified from 4‐weeks‐old Dap5 flox and age‐matched HO‐Dap5 ΔFoxp3 mice and differentiated into iTreg in vitro. The absence of Dap5 protein expression in iTregs from HO‐Dap5 ΔFoxp3 mice was assessed by performing WB. b) Similar spleen sizes between 4‐weeks‐old Dap5 flox and age‐matched HO‐Dap5 ΔFoxp3 mice. c) Bar plots showing unchanged proportions of indicated T cell subpopulations in the PBMC from 4‐weeks‐old HO‐Dap5 ΔFoxp3 and age‐matched Dap5 flox mice. d) Dot plots displaying expression levels of the canonical marker genes across different thymocyte subpopulations. e) Bar plots showing unaltered proportions of Helios − Treg and Helios + Treg in PBMCs, spleen and lymph nodes between 4‐weeks‐old HO‐Dap5 ΔFoxp3 and age‐matched Dap5 flox mice. f) Schematic diagram depicting the workflow of bone morrow (BM) chimera experiment. Briefly, the BM cells were isolated from age‐matched and sex‐matched Dap5 flox (CD45.2), HO‐Dap5 ΔFoxp3 (CD45.2) and wild type (WT) C57BL/6 (CD45.1). The BM cells from HO‐Dap5 ΔFoxp3 or Dap5 flox mice were mixed with BM cells from WT (CD45.1) mice at 1:1 ratio and intravenously injected into lethally irradiated (9 Gy) WT C57BL/6 recipient mice for subsequent bone marrow reconstitution. g) Bar plots comparing the contributions of Helios + Tregs and Helios − Tregs derived from donors of Dap5 flox and HO‐Dap5 ΔFoxp3 mice in the BM‐reconstituted recipients. p‐ values were determined by two‐tailed student's T ‐test (c, e and g), * p <0.05.

Article Snippet: To dissect the function of Dap5 in Tregs in vivo, we generated transgenic mice with specific Dap5 ablation in Tregs ( Dap5 ΔFoxp3 ) by crossing the floxed‐ Dap5 mice with the Foxp3 ‐ Cre (B6.129(Cg)‐ Foxp3 4(YFP/icre)Ayr /J) strain from Jackson Laboratory that are widely used in Treg studies (Figure , Supporting Information).

Techniques: In Vivo, Purification, In Vitro, Expressing, Marker, Isolation, Injection, Irradiation, Derivative Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting DAP5 Disrupts Alternate Mode of Translational Initiation in Tregs and Potentiates Antitumor Immunity

doi: 10.1002/advs.202520625

Figure Lengend Snippet: HO‐ Dap5 ΔFoxp3 mice displayed lethal autoimmune tolerance defects. a) Under the same housing condition, the lifespan of HO‐ Dap5 ΔFoxp3 mice were significantly shortened compared with Dap5 flox littermates. b) Around 6‐8 weeks‐old, HO ‐Dap5 ΔFoxp3 mice had enlarged lymphoid organs, scaly patches on the skin tissues of the head and ears, hair loss, and swollen toes. c) Left panel : 2D uniform manifold approximate projection (2D‐UMAP) plots comparing the distribution of T cell subpopulations in PBMCs between 6‐weeks‐old Dap5 flox and HO‐ Dap5 ΔFoxp3 mice; right panel : stacked bar plots comparing the compositions of naïve T cells and effector T cells between Dap5 flox and HO‐ Dap5 ΔFoxp3 mice. d) Enhanced TCR clonal expansion (left) and reduced Shannon diversity indexes of TCRs (right) in the peripheral T cells from HO‐ Dap5 ΔFoxp3 mice compared with that from Dap5 flox mice. e) 2D‐UMAP plots showing elevated presence of Il4 + basophils in the peripheral blood of HO‐ Dap5 ΔFoxp3 mice. f) Bar plots comparing the frequencies of indicated immune cell populations in the peripheral blood from 6‐weeks‐old HO‐Dap5 ΔFoxp3 mice and age‐matched Dap5 flox mice. g) 2D‐UMAP plots displaying reduced B cell presence in the peripheral blood of HO‐Dap5 ΔFoxp3 mice. h) Reduced BCR clonal expansion (left) and increased Shannon diversity indexes of BCR (right) in the peripheral B cells from HO‐ Dap5 ΔFoxp3 mice compared with that from Dap5 flox mice. i) The bar plot demonstrates a reduction of peripheral B cells in HO‐Dap5 ΔFoxp3 mice relative to the age‐matched Dap5 flox mice. j) The multi‐color immunofluorescence (mIF) pictures showing impaired GC architectures and reduced B cells numbers in the spleens from 6‐weeks‐old HO‐Dap5 ΔFoxp3 mice compared with that from age‐matched Dap5 flox mice. DAPI (blue), CD3 (green), CD19 (red). k) bar plot indicating decreased frequencies of peripheral Treg cells in HO‐Dap5 ΔFoxp3 mice compared to Dap5 flox mice. l) Bar plots showing pTregs, but not CD4 + T or CD8 + T cells, derived from derived from HO‐Dap5 ΔFoxp3 mice were apoptotic (PI + annexin V + ). m) Naïve CD4 + T cells from Dap5 flox or HO‐Dap5 ΔFoxp3 mice were intravenously injected into Rag1 −/− mice. Approximately 20 days later, enlarged spleens and lymph nodes were observed in HO‐Dap5 ΔFoxp3 mice. n) The survival curve showing that the lifespan of Rag1 −/− mice receiving intravenous injection of naïve CD4 + T cells from HO‐Dap5 ΔFoxp3 mice was significantly shortened. p‐ values were determined by Log‐rank test (a and n), or two‐tailed student's T ‐test (f, g, h, i, k and l), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: To dissect the function of Dap5 in Tregs in vivo, we generated transgenic mice with specific Dap5 ablation in Tregs ( Dap5 ΔFoxp3 ) by crossing the floxed‐ Dap5 mice with the Foxp3 ‐ Cre (B6.129(Cg)‐ Foxp3 4(YFP/icre)Ayr /J) strain from Jackson Laboratory that are widely used in Treg studies (Figure , Supporting Information).

Techniques: Immunofluorescence, Derivative Assay, Injection, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting DAP5 Disrupts Alternate Mode of Translational Initiation in Tregs and Potentiates Antitumor Immunity

doi: 10.1002/advs.202520625

Figure Lengend Snippet: Mice with heterozygous Dap5 deletion in Tregs displayed unaltered peripheral immune homeostasis but enhanced Teff response against subcutaneous tumors. a) WB results showing intermediate expression level of Dap5 in pTregs from HE‐ Dap5 ΔFoxp3 mice. b) Bar plot displaying unaltered splenic Treg frequencies in HE‐ Dap5 ΔFoxp3 mice. c) Curves of body weight changes for HE‐ Dap5 ΔFoxp3 and Dap5 flox mice under DSS‐induced colitis modeling. d) Curves of body weight changes for Rag1 −/− mice receiving adoptive transfer of naïve CD4 + T cells or naïve CD4 + T cells mixed with Tregs purified from HE‐ Dap5 ΔFoxp3 mice or Dap5 flox mice. e) Suppressed MC38 tumor growth in HE‐ Dap5 ΔFoxp3 mice. f) Curves monitoring MC38 tumor growth in HE‐ Dap5 ΔFoxp3 mice or Dap5 flox mice. g) Suppressed Panc02 tumor growth in HE‐ Dap5 ΔFoxp3 mice. h) Curves monitoring Panc02 tumor growth in HE‐ Dap5 ΔFoxp3 mice or Dap5 flox mice. i) Violin plots comparing gene expressions between tumor‐infiltrating CD4 + Tconv from HE‐ Dap5 ΔFoxp3 and Dap5 flox mice. j) Bar plots comparing infiltrations of IFN‐γ + CD4 + T cells in the MC38 tumors that grew in HE‐ Dap5 ΔFoxp3 and Dap5 flox mice. k) RNA velocity streamlines projected onto the UMAP‐based embedding. Cells were grouped according to their annotations. l) The directed Partition‐based Graph Abstraction (PAGA) graph showing the connectivity of these CD8 + T subpopulations. The edge weights quantify the connectivity between cell groups. m) Projection of RNA velocity streamlines on UMAP, grouped by cell type. n) GSEA plots displaying enriched signaling pathways in tumor‐infiltrating CD8 + T cells from HE‐ Dap5 ΔFoxp3 mice in contrast to that from Dap5 flox mice. o) Bar plots comparing infiltrations of IFN‐γ + CD8 + T and GZMB + CD8 + T cells between HE‐ Dap5 ΔFoxp3 and Dap5 flox mice. p‐ values were determined by two‐tailed student's T ‐test (b‐d, f, h, j and o), * p <0.05, ** p <0.01, **** p <0.001, *** p <0.001.

Article Snippet: To dissect the function of Dap5 in Tregs in vivo, we generated transgenic mice with specific Dap5 ablation in Tregs ( Dap5 ΔFoxp3 ) by crossing the floxed‐ Dap5 mice with the Foxp3 ‐ Cre (B6.129(Cg)‐ Foxp3 4(YFP/icre)Ayr /J) strain from Jackson Laboratory that are widely used in Treg studies (Figure , Supporting Information).

Techniques: Expressing, Adoptive Transfer Assay, Purification, Protein-Protein interactions, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting DAP5 Disrupts Alternate Mode of Translational Initiation in Tregs and Potentiates Antitumor Immunity

doi: 10.1002/advs.202520625

Figure Lengend Snippet: LLPS of DAP5. a) Phylogenetic tree plot showing that DAP5 is highly conserved in eukaryotic species. b) DAP5 domains aligned with the results of predictor of natural disordered regions (PONDR) and net charge per residue (NCPR) analyses. c) LLPS of purified recombinant eGFP‐DAP5 protein was observed in the buffer supplemented with serial concentrations of protein and NaCl under confocal microscopy. d) The fluorescence recovery after photobleaching (FRAP) curve illustrating the dynamic recovery of eGFP‐DAP5 + puncta in HeLa cells stimulated with 50 µM ETO for 12 h. e) HeLa cells were transfected with indicated DAP5 constructs, then exposed to 50 µM ETO for 12 h. Subcellular eGFP‐DAP5 distribution were subsequently visualized under fluorescence microscopy. f) Observation of eGFP‐Dap5 + puncta in iTregs generated from eGFP‐Dap5‐KI mice under confocal microscopy. g) Total RNAs purified from HeLa cells dose‐dependently enhanced the formation of eGFP‐DAP5 + puncta. p‐ values were determined by two‐tailed student's T ‐test (g), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: To dissect the function of Dap5 in Tregs in vivo, we generated transgenic mice with specific Dap5 ablation in Tregs ( Dap5 ΔFoxp3 ) by crossing the floxed‐ Dap5 mice with the Foxp3 ‐ Cre (B6.129(Cg)‐ Foxp3 4(YFP/icre)Ayr /J) strain from Jackson Laboratory that are widely used in Treg studies (Figure , Supporting Information).

Techniques: Residue, Purification, Recombinant, Confocal Microscopy, Fluorescence, Transfection, Construct, Microscopy, Generated, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting DAP5 Disrupts Alternate Mode of Translational Initiation in Tregs and Potentiates Antitumor Immunity

doi: 10.1002/advs.202520625

Figure Lengend Snippet: Transcripts bound by DAP5 undergo active translation in Tregs. a) Schematic diagram exhibiting the workflow for the combined RIP‐seq, Ribo‐seq and RNA‐seq experiments. b) Bubble plots displaying enriched GO and KEGG terms that were obtained by performing enrichment analysis with transcripts bound by DAP5 in iTregs. c) Integrative Genomics Viewer (IGV) tracks showing representative binding peaks of DAP5 that are associated with cell survival, proliferation and immunomodulation in human iTreg cells. d) Bar plots showing enriched presence of transcripts bound by DAP5 in iTregs. For histogram plotting, qRT‐PCR assays were performed with DAP5‐RIP assay elutes. The RIP‐qPCR data analysis was performed according to the ΔΔCt method. [ ⁷⁷ , ⁷⁸ ] Briefly, ΔCt [normalized RIP] = (Ct [RIP]‐(Ct [Input]‐Log 2 (Input Dilution Factor))), % Input = 2 (‐ΔCt[normalized RIP]) . e) Addition of Cy5‐labeled 5′UTR of MCL1 enhanced formation of eGFP‐DAP5 + puncta in the buffer containing 100 mM NaCl, 20 mM Tris‐HCl (pH = 7.5), 5% PEG8000 and 2 µM eGFP‐DAP5. Pictures were taken under confocal microscopy. f) The volcano plot depicting transcripts in iTregs with significantly increased translation efficiencies (TE) compared to naïve CD4 + T cells. TE values were determined by combined analysis of Ribo‐seq and RNA‐seq data. Transcripts meeting the criteria of absolute log 2 TE − iTreg TE − na ï ve CD 4 ≥ 2 and adjusted p‐ value < 0.05 were designated as differentially translated. g) Lower panel : the curves displaying the density distributions of transcripts along their relative TE values ( log 2 TE − iTreg TE − na ï ve CD 4 ) (X‐axis). Transcripts were grouped into three categories: DAP5‐bound and hypo‐m 6 A modified, DAP5‐bound and m 6 A modified, and m 6 A modified only; upper panel : box plots comparing TE values of the aforementioned categories of transcripts within three indicated ranges of relative TE values: log 2 TE − iTreg TE − na ï ve CD 4 <‐1, 1≤ log 2 TE − iTreg TE − na ï ve CD 4 <4.4, 4.4≤ log 2 TE − iTreg TE − na ï ve CD 4 ≤10. h) Bar plots displaying efficiencies of in vitro mouse iTreg differentiation induced by FB23‐2 or STM2457. i) Flow cytometric histograms comparing Ki‐67 expression levels between mouse iTregs induced by FB23‐2 and STM2457. P‐ values were determined by two‐tailed student's T ‐test (d and h) or Kolmogorov‐Smirnov test and Kruskal‐Wallis (g), * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: To dissect the function of Dap5 in Tregs in vivo, we generated transgenic mice with specific Dap5 ablation in Tregs ( Dap5 ΔFoxp3 ) by crossing the floxed‐ Dap5 mice with the Foxp3 ‐ Cre (B6.129(Cg)‐ Foxp3 4(YFP/icre)Ayr /J) strain from Jackson Laboratory that are widely used in Treg studies (Figure , Supporting Information).

Techniques: RNA Sequencing, Binding Assay, Quantitative RT-PCR, Labeling, Confocal Microscopy, Modification, In Vitro, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting DAP5 Disrupts Alternate Mode of Translational Initiation in Tregs and Potentiates Antitumor Immunity

doi: 10.1002/advs.202520625

Figure Lengend Snippet: Dap5 promotes IL2RA and MCL1 translation to maintain ti‐Treg stability and survival. a) Volcano plots showing differentially expressed genes between ti‐Tregs from HE‐ Dap5 ΔFoxp3 or Dap5 flox mice. b) 2D‐UMAP plots displaying reduced Foxp3 expression among ti‐Tregs from HE ‐Dap5 ΔFoxp3 mice. c) Bar plots comparing CD25 expressions in splenic or tumor‐infiltrating CD4 + Foxp3 + T cells between HE‐ Dap5 ΔFoxp3 or Dap5 flox mice. d) Bar plots comparing frequencies of splenic or tumor‐infiltrating CD4 + Foxp3 + T cells between HE‐ Dap5 ΔFoxp3 or Dap5 flox mice. e) Bar plots comparing proportions of splenic or tumor‐infiltrating cleaved‐caspase3 + Tregs between HE‐ Dap5 ΔFoxp3 or Dap5 flox mice. f) Immunoblotting results showing absence of Mcl‐1 expression in iTregs derived from HO‐ Dap5 ΔFoxp3 . g) Construction strategy for Dap5 flox Foxp3 CreERT2 strain. h) Curves monitoring MC38 tumor growth in Dap5 fl/+ Foxp3 CreERT2 and Foxp3 CreERT2 mice. Tamoxifen was administered on the same day as tumor cell inoculation. i‐k) Bar plots comparing proportions of Tregs in CD4 + T cells (i), cleaved caspase‐3 + Tregs in total Tregs (j) and GZMB + CD8 + T in total CD8 + T cells (k) in tumors from Dap5 fl/+ Foxp3 CreERT2 and Foxp3 CreERT2 mice. P‐ values were determined by two‐tailed student's T ‐test (c, f, h, i, j and k) * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: To dissect the function of Dap5 in Tregs in vivo, we generated transgenic mice with specific Dap5 ablation in Tregs ( Dap5 ΔFoxp3 ) by crossing the floxed‐ Dap5 mice with the Foxp3 ‐ Cre (B6.129(Cg)‐ Foxp3 4(YFP/icre)Ayr /J) strain from Jackson Laboratory that are widely used in Treg studies (Figure , Supporting Information).

Techniques: Expressing, Western Blot, Derivative Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting DAP5 Disrupts Alternate Mode of Translational Initiation in Tregs and Potentiates Antitumor Immunity

doi: 10.1002/advs.202520625

Figure Lengend Snippet: Graphical abstract: Dap5 functions as a molecular switch of translation mode in ti‐Tregs. Chronic stresses in the TME trigger ISR, leading to eIF‐2a phosphorylation and a consequent impairment of CDT activity in Tregs. Meanwhile, ti‐Tregs engage Dap5 to sustain alternate mode of translation of CD25 and MCL‐1, which are critical for ti‐Treg lineage stability and survival in the harsh TME.

Article Snippet: To dissect the function of Dap5 in Tregs in vivo, we generated transgenic mice with specific Dap5 ablation in Tregs ( Dap5 ΔFoxp3 ) by crossing the floxed‐ Dap5 mice with the Foxp3 ‐ Cre (B6.129(Cg)‐ Foxp3 4(YFP/icre)Ayr /J) strain from Jackson Laboratory that are widely used in Treg studies (Figure , Supporting Information).

Techniques: Phospho-proteomics, Activity Assay

Journal: bioRxiv

Article Title: ABL Kinases Modulate EZH2 Phosphorylation and Signaling in Metastatic Triple Negative Breast Cancer

doi: 10.1101/2025.02.18.638898

Figure Lengend Snippet: ( A. ) EZH2 and SUZ12 tyrosine phosphorylation is unchanged upon ABL inhibition. SUM159 cells were treated with either DMSO (vehicle control) or ABL001 for 24 hours, after which cells were lysed and an antibody against IgG (control),EZH2,or SUZ12 was added to cell lysates. Tyrosine Phosphorylation was detected using 4G10 pan phosphotyrosine antibody. Actin was used as a loading control in the WCL. (B.) EZH2 phosphorylation mark abundance was profiled from data generated from TNBC clinical samples hosted on the Proteomic Data Commons.( C.) SUM159 cells were treated with ABL kinase inhibitor ABL001 for 24 hours with ABL001. Lysates were prepared from these cells and cells were probed for endogenous levels of phosphorylated forms of EZH2. (D.) Bone metastatic TNBC cells were lentivirally transduced with either a nontargeting shRNA (SCR) or and shRNA directed against both ABL1 and ABL2 (AA). P-EZH2 (T487) levels were assessed via immunoblot. Actin was used as a loading control. (E.) Linear protein structure of EZH2 with T487 indicated. (F.)HEK293T cells were transiently transfected with constructs expressing either pcDNA3.1_3xFlagEzh2: WT, T487A (phosphodeficient mutant), or T487D (phosphomimetic). Cell lysates were prepared and ectopically expressed EZH2 was pulled down using the fab-trap nanobody system recognizing 3xFlag. Interactions with endogenous proteins were assessed as indicated. Vinculin was used as a loading control for the input. (G.) Bone metastatic TNBC cells were treated with 10uM ABL001 for 72 hours afterwhich EZH2 was immunoprecipitated from the whole cell lysate (WCL) and probed for endogenous interactors via immunoblot. Vinculin was used as a loading control. All data represent n=3 independent experiments, with the exception of (B.).

Article Snippet: EV (pN1-eGFP) and ABL2 eGFP (pN1-ABL2-eGFP WT) constructs were previously generated and described previously( , ). pcDNA3.1-3’3xFlag was a gift from Quanfu Ma (Addgene plasmid # 208616; http://n2t.net/addgene:208616 ; RRID:Addgene_208616); pcDNA3.1_3xFlagEzh2 WT was a gift from Thomas Cech (Addgene plasmid # 173717; http://n2t.net/addgene:173717 ; RRID:Addgene_173717)( ) pCMVHA hEZH2 was a gift from Kristian Helin (Addgene plasmid # 24230; http://n2t.net/addgene:24230 ; RRID:Addgene_24230)( ). pGFP FAK and pGFP FAK Y397F were a gift from Kenneth Yamada (Addgene plasmid # 50515; http://n2t.net/addgene:50515 ; RRID:Addgene_50515)( ).

Techniques: Inhibition, Control, Generated, Transduction, shRNA, Western Blot, Transfection, Construct, Expressing, Mutagenesis, Immunoprecipitation