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: Molecular medicine reports

Article Title: miR-218 is downregulated and directly targets SH3GL1 in childhood medulloblastoma.

doi: 10.3892/mmr.2013.1639

Figure Lengend Snippet: Figure 1. miR-218 expression is downregulated and inversely correlated with the expression of SH3GL1 in MB cancer cell lines. (A) qPCR analysis of the miR-218 expression levels in a panel of human MB cells and in normal cerebellum. (B and C) SH3GL1 expression at the mRNA and protein levels in different human MB cell lines and normal cerebellum. MB, medulloblastoma; GAPDH, glyceraldehyde 3‑phosphate dehydrogenase.

Article Snippet: The sequence of the SH3GL1 siRNA was obtained from Origene (SR304356; Rockville, MD, USA).

Techniques: Expressing

Journal: Molecular medicine reports

Article Title: miR-218 is downregulated and directly targets SH3GL1 in childhood medulloblastoma.

doi: 10.3892/mmr.2013.1639

Figure Lengend Snippet: Figure 2. SH3GL1 is a target of miR-218. (A) Predicted miR-218 target sequences in the 3'-UTR of SH3GL1 (SH3GL1-UTR-WT) and mutants containing three mutated nucleotides in the 3'-UTR of SH3GL1 (SH3GL1-UTR-MUT). (B and C) HEK‑293T cells were cotransfected with miR‑218 or anti‑miR‑218 and psicheck2 vector with SH3GL1 3'-UTR, WT or mutated. After 36 h, the luciferase activity was measured. Renilla luciferase activity was normalized to firefly luciferase expression for each sample. Each experiment was performed in triplicate. *P<0.05. (D) Relative miR-218 expression levels in PSFK and UW228 cells stably expressing miR-218 by qPCR. (E and F) qPCR and western blot analysis were performed to detect the expression of SH3GL1 mRNA and protein in PSFK and UW228 cells stably expressing miR-218. UTR, untranslated region; MUT, mutated; WT, wild type; NC, normal cerebellum.

Article Snippet: The sequence of the SH3GL1 siRNA was obtained from Origene (SR304356; Rockville, MD, USA).

Techniques: Plasmid Preparation, Luciferase, Activity Assay, Expressing, Stable Transfection, Western Blot

Journal: Molecular medicine reports

Article Title: miR-218 is downregulated and directly targets SH3GL1 in childhood medulloblastoma.

doi: 10.3892/mmr.2013.1639

Figure Lengend Snippet: Figure 4. SH3GL1 knockdown phenocopies the miR‑218 overexpression in MB cells. (A and B) Expression of SH3GL1, p‑ERK and p‑Jun were detected by western blot analysis in PSFK or UW228 cells following miR-218 overexpression and transfection with siSH3GL1. (C and D) Cell proliferation analysis of transfected PSFK or UW228 cells with siSH3GL1 or NC by MTT assay at 24, 48 and 72 h following transfection. MB, medulloblastoma; NC, normal cerebellum; GAPDH, glyceraldehyde 3‑phosphate dehydrogenase; p‑ERK, phosphorylated‑extracellular signal‑regulated kinases.

Article Snippet: The sequence of the SH3GL1 siRNA was obtained from Origene (SR304356; Rockville, MD, USA).

Techniques: Knockdown, Over Expression, Expressing, Western Blot, Transfection, MTT Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Elucidation of protein interactions necessary for the maintenance of the BCR–ABL signaling complex

doi: 10.1007/s00018-019-03397-7

Figure Lengend Snippet: Nilotinib causes partial dissolution of BCR–ABL signaling complex. a 293T cells were transfected with p190 BCR–ABL, native cell lysates were subjected to ultracentrifugation in the 15–40% sucrose gradient, and collected fractions were analyzed by western blot. The presence of BCR–ABL signal in more than one fraction suggests the existence of complexes of different compositions. Note the various degrees of co-sedimentation of BCR–ABL with p85a-PI3K, GRB2, SHIP2, SHC1, SOS1, SHP2 and cCBL; no co-sedimentation with CRK, CRKL or GAB2 was found. Inhibition of BCR–ABL kinase activity with 100 nM nilotinib resulted in a shift of a fraction of the BCR–ABL complexes towards lighter fractions, suggesting partial dissolution of the BCR–ABL signaling complex. b The western blot analysis of proteins co-sedimenting with BCR–ABL (p85a-PI3K, GRB2 and SHIP2) was quantified as described in “Materials and methods”. Note that portion of GRB2, but not SHIP2 or p85a-PI3K dissociated from the BCR–ABL complex after nilotinib treatment. Data represent a single experiment out of three independent experiments carried out. The fractions containing most of the p190 BCR–ABL are highlighted in red. Phosphorylation (p) at ABL Y412 was used to determine the degree of BCR–ABL inhibition using nilotinib; actin serves as a loading control in total cell lysates used for ultracentrifugation. c Cells were transfected with FLAG-tagged p190 BCR–ABL, V5-tagged GRB2 or SHIP2, treated with nilotinib, and subjected to PLA. The antibodies against protein tags were used in PLA (red); cABL antibody was used to counterstain the transfected cells (green). Cells transfected with BCR–ABL and an empty vector serve as the negative control. Number of PLA dots per cell was calculated and graphed (10–90 percentile). Statistically significant differences were highlighted (Student’s t test with Welch’s correction for unequal variances; *p < 0.05, **p < 0.01). Scale bars, 10 µm

Article Snippet: GRB2 (TP300469), SHC1 (TP304362), STS1 (TP303523), CRKL (TP308129) and cCBL (TP314069) were obtained from Origene, SHIP2 (P09-20G-10) and p85a-PI3K (P31–30H) were obtained from SignalChem.

Techniques: Dissolution, Transfection, Western Blot, Sedimentation, Inhibition, Activity Assay, Phospho-proteomics, Control, Plasmid Preparation, Negative Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Elucidation of protein interactions necessary for the maintenance of the BCR–ABL signaling complex

doi: 10.1007/s00018-019-03397-7

Figure Lengend Snippet: SHIP2 and GRB2 associate with kinase-inactive BCR–ABL. a Scheme of used experimental procedure comprising native lysis, blue-native (BN)-PAGE, SDS-PAGE and western blot. The three members of protein complex are highlighted in color. b Cell lysates of 293T cells transfected with p210 BCR–ABL. KD, kinase-dead BCR–ABL; Y177, BCR–ABL Y177F mutant. The inhibition of BCR–ABL kinase activity by nilotinib is demonstrated by the lack of autophosphorylation (p) at Y412. Actin serves as loading control. No trans, non-transfected cells. c Merged second dimension BN-PAGE blots of cells transfected with BCR–ABL variants. The membranes have been probed sequentially for BCR–ABL, SHIP2 and GRB2, the BCR–ABL/SHIP2/GRB2 complexes are highlighted by yellow box. (D) Quantification of the percentage of bound GRB2 and SHIP2 to the BCR–ABL. Statistically significant differences are highlighted (Student’s t test, **p < 0.01; n.s., not significant). Data are representative of three independent experiments

Article Snippet: GRB2 (TP300469), SHC1 (TP304362), STS1 (TP303523), CRKL (TP308129) and cCBL (TP314069) were obtained from Origene, SHIP2 (P09-20G-10) and p85a-PI3K (P31–30H) were obtained from SignalChem.

Techniques: Lysis, SDS Page, Western Blot, Transfection, Mutagenesis, Inhibition, Activity Assay, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Elucidation of protein interactions necessary for the maintenance of the BCR–ABL signaling complex

doi: 10.1007/s00018-019-03397-7

Figure Lengend Snippet: The BCR–ABL signaling complex is preserved after nilotinib treatment. 293T cells were transfected with p190 and p210 BCR–ABL alone (a) or together with STS1 (b), CRKL (c) and GRB2 (d). BCR–ABL was immunoprecipitated (IP) and binding of interaction partners was analyzed by western blot. The SHC1 isoforms are indicated (p46, p52, p66). Empty, transfection with empty plasmid. BCR–ABL kinase activity was determined by detecting autophosphorylation (p) at Y412. Note the co-immunoprecipitation of SOS1, SHIP2, cCBL, SHC1, STS1, CRKL and GRB2 with BCR–ABL in cells treated with nilotinib (green arrows). Also note the co-immunoprecipitation of STS1, CRKL, and GRB2 with kinase-dead (KD) BCR–ABL (blue arrows). Data are representative of three independents experiments (n = 3). Actin serves as a loading control in cell lysates used for IP

Article Snippet: GRB2 (TP300469), SHC1 (TP304362), STS1 (TP303523), CRKL (TP308129) and cCBL (TP314069) were obtained from Origene, SHIP2 (P09-20G-10) and p85a-PI3K (P31–30H) were obtained from SignalChem.

Techniques: Transfection, Immunoprecipitation, Binding Assay, Western Blot, Plasmid Preparation, Activity Assay, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Elucidation of protein interactions necessary for the maintenance of the BCR–ABL signaling complex

doi: 10.1007/s00018-019-03397-7

Figure Lengend Snippet: Interaction of GRB2, SOS1, cCBL, and SHC1 with BCR–ABL. a Secondary structure prediction of p210 BCR–ABL by IUPRED. Values above 0.5 indicate disordered regions IDR1 and IDR3 on the BCR–ABL N- and C-termini, involving Y177 and three NLS, respectively. Smaller disordered region IDR2 is located between domains PH and SH3. b Scheme of the microarray analysis. Thirteen amino acid long peptides corresponding to the primary sequence of p210 BCR–ABL were spotted on microarrays, incubated with protein of interest, primary and fluorescently labeled secondary antibodies, and scanned. Fluorescence intensity values for each spot were used to indicate the binding of protein to BCR–ABL peptides. c Microarrays indicate direct binding of GRB2 to phosphorylated Y177. Red lines on BCR–ABL scheme indicate potential binding sites. Graph shows averaged relative intensities for phosphorylated (red) and non-phosphorylated peptides involving peptides with Y177. Error bars indicate SD from three technical replicates shown in Fig. S2. d Co-immunoprecipitation (Co-IP) of BCR–ABL with GRB2 after expression in 293T cells; Y177F substitution abrogates GRB2 association with BCR–ABL as well as deleting the region (construct BT, bottom arrows). Side arrows indicate electrophoretic mobility shift GRB2 phosphorylated by BCR–ABL. e, f Co-immunoprecipitation of endogenous cCBL, SHC1 and SOS1 with transfected BCR–ABL in 293T cells. Please note the compromised SHC1 binding on BCR–ABL–BT, -ΔST, -ΔTK and KD variants (blue arrows). Y177F abrogates binding of SOS1 and largely limits the binding of cCBL (green arrows). Data are representative of three independent experiments (n = 3). g Quantification of SHC1 co-IP with BCR–ABL constructs from (f). SHC1 was normalized to BCR–ABL levels, error bars indicate SD from four independent experiments. Statistically significant differences are indicated (Student’s t test, *p < 0.05, ***p < 0.001; ns non-significant). h Scheme of the proposed interaction. GRB2 binds directly to phosphorylated Y177 and recruits SOS1. cCBL also requires GRB2 for recruitment. SHC1 requires TK domain and pleckstrin homology (PH) domain for binding

Article Snippet: GRB2 (TP300469), SHC1 (TP304362), STS1 (TP303523), CRKL (TP308129) and cCBL (TP314069) were obtained from Origene, SHIP2 (P09-20G-10) and p85a-PI3K (P31–30H) were obtained from SignalChem.

Techniques: Microarray, Sequencing, Incubation, Labeling, Fluorescence, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Construct, Electrophoretic Mobility Shift Assay, Transfection

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: misPLA analyses of phosphorylation states and protein-protein interactions in SK-BR cells with or without EGF stimulation. A) Phosphorylation targets: SK-BR cells were analyzed for phosphorylation of STAT5a (pSTAT5a), STAT3 (pSTAT3), AKT (pAKT), ERK (pERK), and EGFR (pEGFR), under unstimulated and EGF-stimulated conditions. All targets were visualized three at a time in sequential detection cycles and are shown simultaneously (upper panels, “All targets”) and subsequently as individual channels. PLA signals (red, cyan, green, purple) reflect activated protein states detected via dual-recognition proximity ligation. DAPI (blue) labels nuclei. Scale bars = 50 µm. B) Protein-protein interactions: Visualization of the following protein-protein interactions investigated by misPLA in unstimulated and EGF-stimulated SK-BR cells: MEK1–ERK2, GRB2–MEK1, EGFR–GRB2, STAT3–STAT5a, JAK1–JAK3, JAK1–PI3K, JAK2–STAT5a, JAK1–STAT3, and JAK2–JAK3. Upper panels (“All targets”) represent simultaneous visualization of all targets, imaged three at a time in sequential detection cycles, followed by separated signals per interaction. Scale bars = 50 µm.

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques: Phospho-proteomics, Protein-Protein interactions, Ligation

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: misPLA mapping of signaling interactions in a lymph-node Hodgkin lymphoma, mixed cellularity (right neck); Hodgkin lymphoma, lymphocyte-depleted (neck); Hodgkin lymphoma, lymphocyte-predominant (left neck); Hodgkin lymphoma, mixed cellularity (left neck); and thymoma type B3 (mediastinum). Top row (visualization cycle 1) displays MEK1–ERK2 (FITC), EGFR–GRB2 (Cy5) and GRB2–MEK1 (Cy3N) together with DAPI. Middle row (cycle 2) shows STAT3–STAT5a (FITC), JAK1–JAK3 (Cy5) and JAK1–PI3Kp85 (Cy3N). Bottom row (cycle 3) presents JAK2– STAT5a (FITC), JAK2–JAK3 (Cy5) and JAK1–STAT3 (Cy3N). All nine pairs of antibody-oligonucleotide conjugates were applied and then amplified in a single incubation. The RCA products were revealed using detection oligonucleotides conjugated with three fluorophores in three visualization cycles. A standard three-channel fluorescence microscope was used with identical settings for all three fluorophores. Scale bars, 50 µm.

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques: Amplification, Incubation, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: Analysis of primary blood cells from two patients diagnosed with CML, targeting molecular pathways known to be up-regulated in CML. The experiment used a slightly different oligonucleotide design compared to other experiments reported herein, but with similar performance ( .) A, E) Quantitative analysis of numbers of signals per cell revealed striking differences among individual blood cells. Top and bottom rows represent data for two different CML patients. Visualization cycle 1: phosphoPI3K-AKT1 in Cy3 (red), phosphoAKT1-AKT3 in Cy5 (green), AKT(pan)-AKT2 in FITC (yellow). Cycle 2: JAK2-JAK3 in Cy3 (blue), phosphorylated GRB2 in Cy5 (orange), MEK1-ERK in FITC (purple). Cycle 3: STAT3-phosphoSTAT3 in Cy5 (cyan), STAT3-phosphoSTAT3 in FITC (magenta) and STAT3-STAT5 in Cy3 (lime). The same color coding was used throughout all panels in the figure. B, F) Scatterplots of pairs of detection reactions, serving to visualize correlations across detection pairs (colors as in A, B)). C, G) Visualization of a zoomed-in view of detection reactions per cell. Outline of nuclei are shown in red. D, H) Cell to cell heterogeneity is visualized by overlaying individual detection reactions on the cells - here showing zoomed in region with D) phosphorylated GRB2 (in orange) and (h) STAT3-phosphoSTAT3 (in magenta). Cell nuclei are outlined in red. The raw image data from the full sample, together with detections are available for interactive viewing at https://ulflandegren2025.serve.scilifelab.se .

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

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