Journal: Cell Death Discovery

Article Title: Grb2 interacts with necrosome components and is involved in rasfonin-induced necroptosis

doi: 10.1038/s41420-022-01106-1

Figure Lengend Snippet: A The level of reactive oxygen species (ROS) was detected as described in Materials and Methods following ACHN cells were treated with rasfonin (12 μM) or positive control for 12 h. B ACHN cells were treated with rasfonin (12 μM) in the presence or absence of N-acetyl-L-cysteine (NAC, 0.5 mM) for 12 h; cell viability was analyzed by MTS assay as described in Materials and Methods. C ACHN cells were challenged with rasfonin (6 and 12 μM) for 4 h and then, the total RNA was extracted, reversed, and detected by real-time PCR. D , E ACHN cells were treated with rasfonin (0–12 μM) for 4 h, the cells were lysed and subjected to immunoblotting with the antibodies indicated. Actin was used as a loading control. Data are presented as mean ± SD and are representatives of three independent experiments. Each performed in triplicate, and the data was analyzed by t -test. Single asterisk denotes that the group is statistically different from the control groups ( p < 0.05), whereas double asterisk means ( p < 0.01). The ratios between EGFR, RIP1 or Grb2 and Actin (A) were shown below the blots ( D ). Similar experiments were repeated at least three times.

Article Snippet: Small interference RNA (siRNA) of RIP1 (sc-36426 and L-004445-00), EGFR (sc-29301 and L-003114-00) and Grb2 (sc-29334 and L-019220-00) were obtained from Santa Cruz Biotechnology and Dharmacon along with control siRNA.

Techniques: Positive Control, MTS Assay, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Cell Death Discovery

Article Title: Grb2 interacts with necrosome components and is involved in rasfonin-induced necroptosis

doi: 10.1038/s41420-022-01106-1

Figure Lengend Snippet: A The STRING network analysis of possible interactions among EGFR, Grb2, RIP1 and p62. B–E ACHN cells were exposed to rasfonin (6 or 12 μM) for 6 h, and equal amounts of cell lysates were immunoprecipitated with the mouse monoclonal antibodies of Grb2, p62, EGFR, RIP1 or IgG. Immunoprecipitates were then immunoblotted for the indicated polyclonal (Grb2, p62 and EGFR) or monoclonal (RIP1) antibodies. Similar experiments were repeated twice.

Article Snippet: Small interference RNA (siRNA) of RIP1 (sc-36426 and L-004445-00), EGFR (sc-29301 and L-003114-00) and Grb2 (sc-29334 and L-019220-00) were obtained from Santa Cruz Biotechnology and Dharmacon along with control siRNA.

Techniques: Immunoprecipitation, Bioprocessing

Journal: Cell Death Discovery

Article Title: Grb2 interacts with necrosome components and is involved in rasfonin-induced necroptosis

doi: 10.1038/s41420-022-01106-1

Figure Lengend Snippet: A Following transfection with the control (Mock) or Grb2 siRNA (siGrb2) for 48 h, ACHN cells were treated with rasfonin (12 μM) for 4 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies. B The efficiency of siRNA interference was determined with sample in ( A ). C Schematic representation of the WT Grb2 and mutant DN Grb2. D , E HEK293T cells were transfected transiently with the WT Grb2 or DN Grb2 plasmids for 36 h, and treated with rasfonin (12 μM) for 4 h. Cell lysates were analyzed by immunoblotting with the indicated antibody. Actin was used as a loading control. F The transfection efficiency was detected and acquired by using fluorescence microscope. Similar experiments were repeated at least three times.

Article Snippet: Small interference RNA (siRNA) of RIP1 (sc-36426 and L-004445-00), EGFR (sc-29301 and L-003114-00) and Grb2 (sc-29334 and L-019220-00) were obtained from Santa Cruz Biotechnology and Dharmacon along with control siRNA.

Techniques: Transfection, Control, Western Blot, Mutagenesis, Fluorescence, Microscopy

Journal: Cell Death Discovery

Article Title: Grb2 interacts with necrosome components and is involved in rasfonin-induced necroptosis

doi: 10.1038/s41420-022-01106-1

Figure Lengend Snippet: A–E After transfection with the control (Mock) or Grb2 siRNA (siGrb2) for 48 h, ACHN cells were exposed to 12 μM rasfonin for 6 h, and the total RNA was extracted, reversed, and detected by real-time PCR ( A ); cell lysates were analyzed by immunoblotting with the indicated antibodies, Actin was used as a loading control ( B ); the apoptosis and necrosis induced by rasfonin were determined by flow cytometry ( C and D ). Apoptotic: AV-positive and PI-negative; necrotic: PI-positive. The data are presented as mean ± S.D. from three independent experiments. The double asterisk denotes the group is statistically different from the control groups ( P < 0.01). E Cell lysates in ( B ) were analyzed by immunoblotting with the indicated antibodies, and ratios between cleaved PARP-1 (cPARP-1) and PARP-1, or between cPARP-1 and Actin (A), or between PARP-1 and Actin (A) were shown below the blots. Similar experiments were repeated at least three times. F Schematic mechanism of EGFR/Grb2 in rasfonin-dependent autophagy and necroptosis.

Article Snippet: Small interference RNA (siRNA) of RIP1 (sc-36426 and L-004445-00), EGFR (sc-29301 and L-003114-00) and Grb2 (sc-29334 and L-019220-00) were obtained from Santa Cruz Biotechnology and Dharmacon along with control siRNA.

Techniques: Transfection, Control, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry

Journal: The Journal of experimental medicine

Article Title: GRB2 promotes thymocyte positive selection by facilitating THEMIS-mediated inactivation of SHP1.

doi: 10.1084/jem.20221649

Figure Lengend Snippet: Figure 1. Themis-deficient and Grb2-deficient mice exhibit a similar phenotype. (A) Phenotype of thymocytes (left panels) and splenocytes (right panels) from the indicated mice. Numbers are percentage of cells in the indicated gate. Data shown are representative of at least eight mice of each genotype. Efficient

Article Snippet: Flag-tagged Grb2 was subcloned into pFLAGCMV2 vector by PCR with human cDNA for Grb2 from Addgene.

Techniques:

Journal: The Journal of experimental medicine

Article Title: GRB2 promotes thymocyte positive selection by facilitating THEMIS-mediated inactivation of SHP1.

doi: 10.1084/jem.20221649

Figure Lengend Snippet: Figure 2. TCR signaling responses are impaired and SHP1 tyrosine phosphatase activity is increased in Grb2-deficient thymocytes. (A and B) CD3+CD4 antibody activation of DP enriched +/+, Themis−/−, and CD2-iCre;Grb2f/f thymocytes. Thymocytes were rested for 6 h at 37°C, pre-incubated with biotinylated anti- CD3 and anti-CD4 antibodies, then streptavidin was added for the indicated times at 37°C. Cells were lysed at 4°C and proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes then blotted with the indicated antibodies. Results shown are representative of three experiments. (C) Oxidized SHP1 in Themis−/−and CD2-iCre;Grb2f/f thymocytes following treatment with H2O2. SHP1 (arrowhead) was identified by its mobility on SDS-PAGE. (D) Summary of experiments as shown in C. +/+ was arbitrarily set to 100% for comparison. Error bars show SD. ***P < 0.005. T test, two tailed, Type 2. Data shown are from four experiments. (E and F) GRB2 and THEMIS cooperate to inhibit SHP1 tyrosine phosphatase activity. HEK-293 cells were transfected with the indicated plasmids and Western blots of cell lysates were performed with the indicated antibodies. Arrowhead designates endogenous GRB2. Source data are available for this figure: SourceData F2.

Article Snippet: Flag-tagged Grb2 was subcloned into pFLAGCMV2 vector by PCR with human cDNA for Grb2 from Addgene.

Techniques: Activity Assay, Activation Assay, Incubation, SDS Page, Comparison, Two Tailed Test, Transfection, Western Blot

Journal: The Journal of experimental medicine

Article Title: GRB2 promotes thymocyte positive selection by facilitating THEMIS-mediated inactivation of SHP1.

doi: 10.1084/jem.20221649

Figure Lengend Snippet: Figure 3. The developmental defect in Grb2-deficient thymocytes is rescued by deletion of Shp1 (Ptpn6) and exacerbated by overexpression of SHP1. (A and B) Phenotype of thymocytes (left panels) and splenocytes (right panels) from the indicated mice. Numbers are percentage of cells in the indicated gate. Data shown are representative of at least four mice of each genotype. Efficient Cre-mediated deletion was confirmed in all cases where appropriate by Western blot.

Article Snippet: Flag-tagged Grb2 was subcloned into pFLAGCMV2 vector by PCR with human cDNA for Grb2 from Addgene.

Techniques: Over Expression, Western Blot

Journal: The Journal of experimental medicine

Article Title: GRB2 promotes thymocyte positive selection by facilitating THEMIS-mediated inactivation of SHP1.

doi: 10.1084/jem.20221649

Figure Lengend Snippet: Figure 4. The developmental defect in OT2 TCR transgenic Grb2-deficient thymocytes is rescued by deletion of Shp1 (Ptpn6) and exacerbated by overexpression of SHP1. (A) Phenotype of thymocytes (left panels) and splenocytes (right panels) from the indicated mice. (B) Phenotype of OT2 TCR transgenic Themis−/−mice and rescue of the developmental defect in OT2 TCR transgenic Themis−/−mice by deletion of Shp1 (Ptpn6) shown for comparison to A. Numbers indicate percentage of cells in the indicated gate. Data shown are representative of at least four mice for each genotype. Efficient Cre-mediated deletion was confirmed in all cases where appropriate by Western blot.

Article Snippet: Flag-tagged Grb2 was subcloned into pFLAGCMV2 vector by PCR with human cDNA for Grb2 from Addgene.

Techniques: Transgenic Assay, Over Expression, Comparison, Western Blot

Journal: The Journal of experimental medicine

Article Title: GRB2 promotes thymocyte positive selection by facilitating THEMIS-mediated inactivation of SHP1.

doi: 10.1084/jem.20221649

Figure Lengend Snippet: Figure 5. Deletion of Ptpn6 (Shp1) alleviates the signaling defect in GRB2-deficient thymocytes. CD3+CD4 antibody activation of +/+, CD2-iCre;Grb2f/f,

Article Snippet: Flag-tagged Grb2 was subcloned into pFLAGCMV2 vector by PCR with human cDNA for Grb2 from Addgene.

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: EGF Stimulates Rab35 Activation and Gastric Cancer Cell Migration by Regulating DENND1A-Grb2 Complex Formation

doi: 10.3389/fphar.2018.01343

Figure Lengend Snippet: DENND1A and Grb2 combine with each other. (A) BGC-823 cells lysates were immunoprecipitated with an anti-Grb2 antibody or (B) anti-DENND1A antibody, and then both unprocessed lysates (Input) and immunoprecipitates were analyzed by western blot with indicated antibodies. (C) HEK-293T cells transiently transfected with Flag-Grb2 and HA-DENND1A were immunoprecipitated with an anti-HA antibody or anti- Flag antibody, and then both unprocessed lysates (Input) and immunoprecipitates were analyzed by western blot with indicated antibodies. (D) GST-Grb2 was incubated in vitro with lysates of HEK-293T cells transfected with or without HA-DENND1A, and coprecipitation of DENND1A with GST-Grb2, bound to glutathione-beads, was analyzed by western blot. GST, GST alone used as a control. (E) BGC-823 cells expressing Flag-Grb2 and HA-DENND1A were cultured on coverslips and fixed and double-stained with anti-HA and anti-Flag antibody. (F) Original colors were Flag-Grb2, green; HA-DENND1A, red; and nuclei, blue. Merged pictures represent the composite of all channels, with yellow regions indicative of colocalization. Scale bar, 10 μm. All experiments were repeated at least three times.

Article Snippet: Anti-human DENND1A monoclonal antibody, anti-human Grb2 monoclonal antibody, anti-human HA monoclonal antibody and anti-EGFR monoclonal antibody were purchased from Cell Signaling Technology (Danvers, MA, United States).

Techniques: Immunoprecipitation, Western Blot, Transfection, Incubation, In Vitro, Control, Expressing, Cell Culture, Staining

Journal: Frontiers in Pharmacology

Article Title: EGF Stimulates Rab35 Activation and Gastric Cancer Cell Migration by Regulating DENND1A-Grb2 Complex Formation

doi: 10.3389/fphar.2018.01343

Figure Lengend Snippet: DENND1A binds to the SH3 domain of Grb2 through PRD. (A) Schematic diagram of different Flag-tagged Grb2 fragments and HA-tagged DENND1A fragments. (B) Coprecipitation of HA-tagged DENND1A fragments or (C) Flag-tagged Grb2 fragments with Flag-Grb2 or HA-DENND1A were analyzed by immunoblotting. Unprocessed lysates of HEK-293T cells transfected with Flag-Grb2 or HA-DENND1A was used as input. All experiments were repeated at least three times.

Article Snippet: Anti-human DENND1A monoclonal antibody, anti-human Grb2 monoclonal antibody, anti-human HA monoclonal antibody and anti-EGFR monoclonal antibody were purchased from Cell Signaling Technology (Danvers, MA, United States).

Techniques: Western Blot, Transfection

Journal: Frontiers in Pharmacology

Article Title: EGF Stimulates Rab35 Activation and Gastric Cancer Cell Migration by Regulating DENND1A-Grb2 Complex Formation

doi: 10.3389/fphar.2018.01343

Figure Lengend Snippet: EGF stimulation promotes EGFR recruitment of Grb2-DENND1A complex. (A) BGC-823 cells transfected with Flag-Grb2 and HA-DENND1A were treated with or without EGF. Cells were lysed and analyzed for coprecipitation of DENND1A and Grb2 by western blot. Cells transfected with Flag-Grb2 and HA-DENND1A were used as input. (B) BGC-823 cells transfected with Flag-Grb2 were treated with or without EGF. Cells were lysed and analyzed for coprecipitation of Grb2 and EGFR by western blot. Cells transfected with Flag-Grb2 were used as input. (C) BGC-823 cells were treated with or without EGF and fixed and double-stained with anti-DENND1A and anti-EGFR antibody. Original colors were DENND1A or EGFR, red and nuclei, blue. Merged pictures represent the composite of both channels. Scale bar, 10 μm. All the experiments were repeated at least three times.

Article Snippet: Anti-human DENND1A monoclonal antibody, anti-human Grb2 monoclonal antibody, anti-human HA monoclonal antibody and anti-EGFR monoclonal antibody were purchased from Cell Signaling Technology (Danvers, MA, United States).

Techniques: Transfection, Western Blot, Staining

Journal: Frontiers in Pharmacology

Article Title: EGF Stimulates Rab35 Activation and Gastric Cancer Cell Migration by Regulating DENND1A-Grb2 Complex Formation

doi: 10.3389/fphar.2018.01343

Figure Lengend Snippet: The binding of Grb2 and DENND1A affects the activity of Rab35 and the migration of BGC823 cells. (A) BGC-823 cells transfected with or without Flag-Grb2-ΔSH3-N-C were treated with or without EGF. The expression of endogenous activated and total Rab35 was analyzed by western blot. (B) And, the relative migration rate was calculated by normalizing the values obtained for the group of BGC-823 cells treated without Flag-Grb2-ΔSH3-N-C and EGF at 24 h as 100%. (C) Transwell assay was performed on BGC-823 cells transfected with or without Flag-Grb2-ΔSH3-N-C to evaluate the ability of migration and invasion. All experiments were repeated at least three times ( ∗∗ P < 0.01; ∗∗∗ P < 0.001).

Article Snippet: Anti-human DENND1A monoclonal antibody, anti-human Grb2 monoclonal antibody, anti-human HA monoclonal antibody and anti-EGFR monoclonal antibody were purchased from Cell Signaling Technology (Danvers, MA, United States).

Techniques: Binding Assay, Activity Assay, Migration, Transfection, Expressing, Western Blot, Transwell Assay

Journal: Biochemistry

Article Title: Recruitment of the adaptor protein Grb2 to EGFR tetramers.

doi: 10.1021/bi500182x

Figure Lengend Snippet: Figure 1. Proteins (EGF, EGFR, and Grb2), EGFR component states, and protein−protein interfaces considered in our computational model. The EGFR ectodomain is taken to be free or bound to EGF. A cytoplasmic domain of EGFR, comprising the juxtamembrane region (JM) and kinase domain, is taken to be locked (i.e., unavailable for interaction) or freed (i.e., available for interaction). The C-terminal tail of EGFR is taken to contain, as a simplification, a single docking site for Grb2, which can be unphosphorylated (Y) and inactive or phosphorylated (pY) and active.

Article Snippet: Human Grb2 (growth factor receptor-bound protein-2) transcript variant 1, as 10 μg of transfection-ready DNA, was purchased from OriGene (catalog no. SC111933) in the vector pCMV6-XL5.

Techniques:

Journal: Biochemistry

Article Title: Recruitment of the adaptor protein Grb2 to EGFR tetramers.

doi: 10.1021/bi500182x

Figure Lengend Snippet: Figure 2. Illustration of the rules for interactions in our computational, rule-based model. The model, which captures the mass-action chemical kinetics of the indicated interactions, consists of 16 rules, which are either reversible (and associated with two rate constants) or unidirectional (and associated with a single rate constant). Each rule represents an interaction. The glyphs used here to represent proteins and protein components are the same as those presented in Figure 1. Here, in illustrating a rule, we use a question mark (?) to indicate a missing protein component or component state that is not depicted explicitly; the missing component or state is taken to have zero influence on the interaction represented by the rule. Similarly, representation of an EGFR ectodomain by a dotted triangle is meant to indicate that the ectodomain may or may not be present in a complex, without influence on the interaction of concern. The model is the same as that presented in our earlier report25 except that a rule for Grb2 binding to phosphorylated EGFR has been added. This rule is illustrated in the lower left box.

Article Snippet: Human Grb2 (growth factor receptor-bound protein-2) transcript variant 1, as 10 μg of transfection-ready DNA, was purchased from OriGene (catalog no. SC111933) in the vector pCMV6-XL5.

Techniques: Binding Assay

Journal: Biochemistry

Article Title: Recruitment of the adaptor protein Grb2 to EGFR tetramers.

doi: 10.1021/bi500182x

Figure Lengend Snippet: Figure 4. FLIM data of individual living BaF/3 cells represented on a phasor diagram. Data points correspond to BaF/3 cells transfected with EGFR−eGFP alone (blue diamond), EGFR−eGFP + EGF (second blue diamond), EGFR−eGFP/Grb2−mRFP (red-filled diamond), EGFR−eGFP/Grb2−mRFP + EGF (red-filled triangle), and untransfected control cells (blue filled circle). Blue solid line denotes trajectory for mixtures of background and EGFR−eGFP. Red line indicates trajectory for EGFR−eGFP/Grb2−mRFP FRET complex mixing with background and EGFR−eGFP fluorescence.

Article Snippet: Human Grb2 (growth factor receptor-bound protein-2) transcript variant 1, as 10 μg of transfection-ready DNA, was purchased from OriGene (catalog no. SC111933) in the vector pCMV6-XL5.

Techniques: Transfection, Control

Journal: Biochemistry

Article Title: Recruitment of the adaptor protein Grb2 to EGFR tetramers.

doi: 10.1021/bi500182x

Figure Lengend Snippet: Figure 3. FLIM data of living BaF/3 cell populations represented on a phasor diagram. (A) Phasor diagram over a limited data range. (B) Phasor diagram on an expanded scale. Individual data points represent the cell-phasor components [x = m cos(φ); y = m sin(φ)] averaged from >20 cells. Data points correspond to BaF/3 cells transfected with EGFR−eGFP alone (blue diamond), EGFR−eGFP + EGF (second blue diamond), EGFR−eGFP/Grb2−mRFP (red-filled diamond), EGFR−eGFP/Grb2−mRFP + EGF (red-filled triangle), and un- transfected control cells (blue filled circle).

Article Snippet: Human Grb2 (growth factor receptor-bound protein-2) transcript variant 1, as 10 μg of transfection-ready DNA, was purchased from OriGene (catalog no. SC111933) in the vector pCMV6-XL5.

Techniques: Transfection, Control

Journal: Biochemistry

Article Title: Recruitment of the adaptor protein Grb2 to EGFR tetramers.

doi: 10.1021/bi500182x

Figure Lengend Snippet: Figure 5. FRET−FLIM−ICS on living BaF/3 cells cotransfected with EGFR−eGFP and Grb2−mRFP. (A) Fluorescence image of EGFR− eGFP/Grb2−mRFP complexes. (B) Spatial autocorrelation image of EGFR−eGFP/Grb2−mRFP complexes. (C) Density of Grb2−mRFP- bound EGFR−EGFP clusters as a function of the density of Grb2− free EGFR−eGFP clusters. The solid line is fit to a Hill function (CDbound = A/(1 + ((Kd/CDfree)(N−1)), with N = 4.1, A = 27, and Kd = 18 clusters).

Article Snippet: Human Grb2 (growth factor receptor-bound protein-2) transcript variant 1, as 10 μg of transfection-ready DNA, was purchased from OriGene (catalog no. SC111933) in the vector pCMV6-XL5.

Techniques: Fluorescence

Journal: Biochemistry

Article Title: Recruitment of the adaptor protein Grb2 to EGFR tetramers.

doi: 10.1021/bi500182x

Figure Lengend Snippet: Figure 6. (A) Plot of simulation results depicting the cluster distribution of Grb2-bound EGFR as a function of EGF concentration. Note that at all concentrations of EGF the EGFR tetramer is the predominant form associated with Grb2. The curves corresponding to dimer and trimer are indistinguishable from monomer because the total number of these oligomeric forms bound to Grb2 is almost negligible. (B) Cluster size distribution of EGFR bound to Grb2 and unbound (free) to Grb2 from simulation with 10 nM EGF.

Article Snippet: Human Grb2 (growth factor receptor-bound protein-2) transcript variant 1, as 10 μg of transfection-ready DNA, was purchased from OriGene (catalog no. SC111933) in the vector pCMV6-XL5.

Techniques: Concentration Assay

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 1. GRB2 complexes with AGO2 under non-stimulated conditions. Schematic diagram of, (a) AGO2 and, (b) GRB2 domain structures. Domains are named and colour coded and attributed amino acid sequence number. Red arrows indicate positions of PXXP motifs investigated in this work. (c) Western blot of AGO2 co-immunoprecipitated with GRB2 in serum starved HEK293T, A498 and PC3 cells. A longer exposure was used to capture AGO2 bands than for GRB2 and GAPDH. All images are taken from the same western blot. (d) Fluorescence and fluorescence resonance energy transfer signals of RFP-tagged GRB2 and GFP-tagged AGO2. HEK293T cells overexpressing fluorescent proteins were serum-starved before imaging. N = 3. Scale bars are 10 μm.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Sequencing, Western Blot, Immunoprecipitation, Fluorescence, Förster Resonance Energy Transfer, Imaging

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 2. Binding of GRB2 to AGO2 is mediated by GRB2 NSH3 and a PXXP motif in AGO2 PAZ domain. (a) Isothermal titration calorimetry (ITC) of a peptide spanning the proline-rich motif 323PHLP326 in AGO2 PAZ domain. (KD = 4.27 ± 1.17 µM). (b, c) ITC of MBP-tagged AGO2 PAZ domain titrated into GRB2. (b) PAZ WT (KD = 585 ± 61 nM). (c) No binding observed for mutation of PXXP (MBP-PAZ 4A). N = 2. (d) Fluorescence resonance energy transfer (FRET) between wild type (WT) and 323AAAA326 (4A) mutant GFP-tagged AGO2 and RFP-tagged GRB2 in HEK293T cells under conditions of serum starvation. White arrows indicate intracellular puncta which show increased FRET when WT AGO2 is expressed. N = 2. Scale bars are 10 μm. (e) Fluorescence lifetime imaging microscopy of RFP-tagged GRB2 proteins and GFP-AGO2 overexpressed in serum-starved HEK293T cells. The formation of a protein complex results in a reduction in fluorescent lifetime represented by a shift to the left of the population of fluorophores (measured in number of pixels). Lifetime population distribution shown by red line on graphs. x = Lifetime (ns), y = number of pixels. Solid black line corresponds to average fluorescent lifetime for GFP, 2.1 ns. Scale bars 25 μm. (f) Expanded region of interest (ROI) further exemplifying left-shift for AGO2/NSH3-SH2 interaction.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Binding Assay, Isothermal Titration Calorimetry, Mutagenesis, Fluorescence, Förster Resonance Energy Transfer, Imaging, Microscopy

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 3. Impact of GRB2-AGO2 complex on interaction with DICER1 and miRNA. (a) Western blot of AGO2 and DICER1 pulldown by GST-GRB2 in HEK293T cells. HEK293T cells were serum-starved before lysis. Bands captured with both a long and short exposure are shown for DICER1, whereas only the image captured with a short exposure is shown for AGO2. GST proteins were detected by ponceau stain. All images are taken from the same western blot. N = 3. (b–d) MST of AGO2 binding to DICER1 C-terminal region, upon pre-incubation of AGO2 with increasing concentrations of GRB2. The difference in binding affinity was negligible. (e) MST of GRB2 with DICER1 C-terminal region. No binding is observed within a physiologically relevant range hence the two do not interact directly. (f) Expanded ribbon model of molecular docking of GRB2 (green; PDB: 1GRI77) to AGO2 PAZ domain (cyan; red and blue indicate positive and negative charges respectively; PDB: 6RA478). The 323PHLP326 sequence is shown (yellow). GRB2 W36 (magenta) interacts with AGO2 P249 (red). Other residues in GRB2 which may contribute towards the interaction are shown in orange. Also shown is space-filling representation of AGO2 PAZ domain with PRM shown (below); and ribbon model of PAZ domain rotated by 90° to highlight juxtaposition of GRB2 binding site PRM and docking site for miRNA (right). Figures generated using PyMOL.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Western Blot, Lysis, Staining, Binding Assay, Incubation, Sequencing, Generated

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 4. GRB2 regulates miRNA expression in HEK293T cells. (a) Western blot of GRB2 expression in wild type (293 T) and depleted (G1) HEK293T clones 1 (G1.1) and 2 (G1.2). While G1.1 is a complete knockout, G1.2 contains a deletion and large insertion in the N-terminal SH3 domain. GRB2 was blotted with an antibody which recognised the C-terminal SH3 domain. Both long and short exposures were used to capture the GRB2 bands, whereas the GAPDH image was captured using a short exposure only. All images are taken from the same western blot. N = 3. (b) Heat plot highlighting miRNAs which show significant log2(fold changes) in expression (p < 0.05) between wild type HEK293T and G1 cells, measured by small RNA sequencing. Cells were deprived of growth factor. miRNAs demonstrated positive (red) and negative (blue) expression changes. N = 2. (c, d) RT-qPCR analysis of fold-change in mean expression of precursor miRNA transcripts (precursor and primary, pre-mir-, hashed bars) and mature miRNA (miR-, plain bars) derived from serum-starved G1 or wild type HEK293T cells. Two groups of miRNAs were observed: (c) miRNAs which diminished at both the level of the precursor and mature transcripts and, (d) miRNAs which were enhanced as mature transcripts but not as precursors. Comparisons were made using a two-tailed Student’s t-test and error bars show standard error of mean. N = 4. ns = not significant.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Expressing, Western Blot, Clone Assay, Knock-Out, RNA Sequencing, Quantitative RT-PCR, Derivative Assay, Two Tailed Test

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 5. The GRB2-let-7 axis regulates oncogene expression. (a) RT-qPCR measurement of fold change in mean expression of let-7 g-5p miRNA and five target mRNAs in serum-starved GRB2 knockout cells (G1) compared to wild type HEK293T (293 T). Comparisons were made using a two-tailed Student’s t-test and error bars show standard error of mean. N = 3. (b) Western blot and (c) quantification of mean protein expression of let-7 targets in growth-factor-deprived G1 and HEK293T cells. The higher molecular band detected by the GRB2 antibody in G1 corresponds to an NSH3-mutated GRB2 polypeptide. For blot 1, a longer exposure was used to capture the DICER1 and GRB2 bands than was used for LIN28B and α-Tubulin. For blot 2, HMGA2 bands were captured using a longer exposure than that required for GRB2 and GAPDH. (d) Quantification of the area covered by migration of HEK293T cells expressing GFP-tagged wild type AGO2 (WT) or an AGO2 mutant which is incapable of binding GRB2 (4A), under conditions of reduced growth factor. Comparisons were made using a two-tailed Student’s t-test and error bars show standard error of mean. N = 3.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Expressing, Quantitative RT-PCR, Knock-Out, Two Tailed Test, Western Blot, Migration, Mutagenesis, Binding Assay

Journal: Oncogene

Article Title: Bcr phosphorylated on tyrosine 177 binds Grb2.

doi: 10.1038/sj.onc.1201053

Figure Lengend Snippet: Figure 5 Anti-Bcr immune complexes contain Grb2 from COS1 cells coexpressing P160 BCR and P145 ABL. (a) Western blotting of anti-BCR(1256 ± 1271) immunoprecipitates from COS1 extracts with anti-BCR (7C6, Santa Cruz Biotechnology, Santa Cruz, CA). The bands were detected using the ECL method. (b) Phosphotyrosine 177 of P160 BCR is critical for its interaction with the simian Grb2 protein. Anti-Grb2 Western blotting was performed on anti-BCR(1256 ± 1271) antibody immunoprecipi- tates; the lanes are as marked in panel a. (c) Phosphotyrosine 177 of P160 BCR is critical for binding the Grb2 SH2 domain. COS1 extracts were incubated with GST-SH2 (Grb2) and the bound proteins were analysed by Western blotting with anti-BCR (7C6, Santa Cruz). Lanes are as marked in panel a. (d) Western blotting of COS1 lysates with anti-Abl. The lysates from COS1 cells were Western blotted with anti-Abl 8E9 monoclonal antibody. The lanes are as marked in panel a

Article Snippet: We and others demonstrated that tyrosine phosphorylation of Bcr sequences within Bcr-Abl by the constitutively active tyrosine kinase of Bcr-Abl represents a pathological phenomenon in pH-positive 1 2 3 4 5 6 C O S 1 B C R (F 17 7) B C R B C R (F 17 7) & A B L B C R & A B L A B L a b c d ➝ ➝ ➝ ➝ P160 Grb2 P160 P145 Figure 5 Anti-Bcr immune complexes contain Grb2 from COS1 cells coexpressing P160 BCR and P145 ABL. (a) Western blotting of anti-BCR(1256 ± 1271) immunoprecipitates from COS1 extracts with anti-BCR (7C6, Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Western Blot, Binding Assay, Incubation

Journal: Oncogene

Article Title: Bcr phosphorylated on tyrosine 177 binds Grb2.

doi: 10.1038/sj.onc.1201053

Figure Lengend Snippet: Figure 6 Anti-Grb2 immune complexes contain P160 BCR from COS1 cells coexpressing P160 BCR and P145 ABL. (a) Western blotting of anti-Grb2 immunoprecipitates with anti-Bcr(1 ± 16). Lanes are as marked. (b) Western blotting of anti-Grb2 immunoprecipitates with anti-Grb2. Lanes are as marked in panel a

Article Snippet: We and others demonstrated that tyrosine phosphorylation of Bcr sequences within Bcr-Abl by the constitutively active tyrosine kinase of Bcr-Abl represents a pathological phenomenon in pH-positive 1 2 3 4 5 6 C O S 1 B C R (F 17 7) B C R B C R (F 17 7) & A B L B C R & A B L A B L a b c d ➝ ➝ ➝ ➝ P160 Grb2 P160 P145 Figure 5 Anti-Bcr immune complexes contain Grb2 from COS1 cells coexpressing P160 BCR and P145 ABL. (a) Western blotting of anti-BCR(1256 ± 1271) immunoprecipitates from COS1 extracts with anti-BCR (7C6, Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Western Blot