Journal: Scientific Reports

Article Title: GRB2 promotes brain metastasis in HER2-positive breast cancer by regulating the Ras/MAPK pathway

doi: 10.1038/s41598-025-99685-3

Figure Lengend Snippet: Association between GRB2/HER2 expression and prognosis in breast cancer. ( A ) Time to Brain Metastasis (TTBM) in breast cancer patients stratified by ER and HER2 Status. P-value was calculated by Mantel-Cox. **** denotes a P-value < 0.0001. ( B ) Overall Survival After Brain Metastasis (OSBM) in breast cancer patients stratified by ER and HER2 Status. ( C ) Overall Survival (OS) in breast cancer patients stratified by ER and HER2 Status. ( D ) GRB2 is a differentially expressed gene associated with brain metastasis in HER2-Positive breast cancer: Evidence from GSE43837. ( E ) Box plots illustrating differences in GRB2 expression levels between normal breast tissue(N) and breast cancer tissues(T). P-value < 0.001. ( F ) Overall Survival of breast cancer patients stratified by low or high GRB2 protein expression (lighter lines represent 95% confidence intervals). ( G ) Scatter plot of the correlation between GRB2 and HER2 expression. ( H ) Bubble plot of enriched pathways identified by GO: BP analysis. ( I ) Venn diagram illustrating the overlap between the MAPK pathway and the ERBB pathway. ( J ) Protein-protein interaction network analysis of key proteins.

Article Snippet: The antibodies used were a rabbit anti-GRB2 antibody (Bioss, China) and a rabbit anti-ERBB2 antibody (BOSTER, China).

Techniques: Expressing

Journal: Scientific Reports

Article Title: GRB2 promotes brain metastasis in HER2-positive breast cancer by regulating the Ras/MAPK pathway

doi: 10.1038/s41598-025-99685-3

Figure Lengend Snippet: Results of cell proliferation, apoptosis, migration, and invasion across cell Lines. A-B. Expression levels of GRB2 and HER2.* denotes a P-value < 0.05. C-D. Differences in cell proliferation and apoptosis across experimental groups. E. Differences in cell migration and invasion capabilities.* denotes a P-value < 0.05.***denotes a P-value < 0.01. F. Microscopic examination of transwell migration and invasion assays. Blue staining indicates migrated or invaded cells, with a higher cell number reflecting stronger migration or invasion capability. G. Differential expression of proteins in the Ras/MAPK pathway by western blot. β-actin serves as the reference protein band, and the darkness of the target band indicates high expression of the target protein in this sample.

Article Snippet: The antibodies used were a rabbit anti-GRB2 antibody (Bioss, China) and a rabbit anti-ERBB2 antibody (BOSTER, China).

Techniques: Migration, Expressing, Staining, Western Blot

Journal: Scientific Reports

Article Title: GRB2 promotes brain metastasis in HER2-positive breast cancer by regulating the Ras/MAPK pathway

doi: 10.1038/s41598-025-99685-3

Figure Lengend Snippet: GRB2 promotes HER2-positive tumor cells to cross the blood brain barrier via the Ras/MAPK pathway in vivo. ( A ) In situ injection model (model 1). ( B ) Fluorescence intensity analysis using an in vivo animal imaging system.areas of high fluorescence intensity indicate active tumor sites or metastases. ( C ) Measurement of ex vivo tumor size and characteristics. ( D ) Results of Hematoxylin & Eosin (H&E) staining (×400), TUNEL assay, and Immunohistochemistry (IHC). H&E staining: Blue-purple represents nuclear staining, highlighting cell nuclei for morphological analysis. TUNEL assay: Yellow staining indicates apoptotic cells, reflecting DNA fragmentation during apoptosis. Immunohistochemistry: Brown represents a moderate positive signal for the target protein, while yellow indicates a weak positive signal, demonstrating varying levels of target protein expression. ( E ) H&E staining (×400) of Metastatic sites in the Brain, Lung, and Liver. ( F ) H-Score evaluation of GRB2 and HER2 protein expression levels. ( G ) Expression levels of key proteins in the Ras/MAPK pathway by western blot. ( H ) Western blot analysis of key proteins in the Ras/MAPK pathway.

Article Snippet: The antibodies used were a rabbit anti-GRB2 antibody (Bioss, China) and a rabbit anti-ERBB2 antibody (BOSTER, China).

Techniques: In Vivo, In Situ, Injection, Fluorescence, Imaging, Ex Vivo, Staining, TUNEL Assay, Immunohistochemistry, Expressing, Western Blot

Journal: Scientific Reports

Article Title: GRB2 promotes brain metastasis in HER2-positive breast cancer by regulating the Ras/MAPK pathway

doi: 10.1038/s41598-025-99685-3

Figure Lengend Snippet: GRB2 promotes HER2-positive tumor cells to cross the blood brain barrier and metastasize in a retrograde manner in vivo. ( A ) Direct cerebral injection model (Model 2). ( B ) H&E Staining (×400) of metastatic sites in the Brain, Lung, and Liver.

Article Snippet: The antibodies used were a rabbit anti-GRB2 antibody (Bioss, China) and a rabbit anti-ERBB2 antibody (BOSTER, China).

Techniques: In Vivo, Injection, Staining

Journal: Scientific Reports

Article Title: GRB2 promotes brain metastasis in HER2-positive breast cancer by regulating the Ras/MAPK pathway

doi: 10.1038/s41598-025-99685-3

Figure Lengend Snippet: Overlap analysis of RNA sequencing and fRIP-Seq results. ( A ) Heatmap visualization of GRB2 knockdown-induced up- or down-regulation of 5 Ras/MAPK pathway-related genes. ( B ) Heatmap reveals 16 Ras/MAPK signaling pathway-related genes with alternative splicing changes in GRB2 knockdown cells. ( C ) Venn diagram shows 1 overlapped gene between 11 Ras/MAPK pathway-related DEGs and 3196 GRB2-bound peaks genes from fRIP-seq data. ( D ) Venn diagram shows 1 overlapped gene between 16 Ras/MAPK pathway-related RASE and 40,709 GRB2-bound peaks genes from fRIP-seq data. ( E ) GRB2-binding peak genes of MESD. ( F ) GRB2-binding peak genes of KITLG.

Article Snippet: The antibodies used were a rabbit anti-GRB2 antibody (Bioss, China) and a rabbit anti-ERBB2 antibody (BOSTER, China).

Techniques: RNA Sequencing, Knockdown, Alternative Splicing, Binding Assay

Journal: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: a , b Representative images of tumor slices from G600 tumors without (control) or with BCI-121 treatment at a concentration of 300 µM for 7 days ( a ). The MTT assay was performed after harvesting the slices 7 days later and the quantifications of cell viability ( b ) in ( a ) (n = 3 mice/group). The IHC against antibodies of Smyd3, H3K4me3, Ki67 ( c ) and quantifications of protein intensities ( d ) from the same cohort of tumor slices in ( a , b ) (n = 3 mice/group). Scale bar is 50 μm. Representative tumor images from nude mice implanted with G600 parental and sgSmyd3-G600 ( e ) and sgShcbp1-G600 ( f ) in mammary fat pads with 2 × 10 5 cells per mouse for 27 days (n = 10-12 mice/group). The tumor weight plots from the same cohort of mice with expression of sgSmyd3 ( g ) or sgShcbp1 ( h ). The protein levels of Smyd3, Shcbp1, Kras, pMek, and pErk in tumors initiated with parental G600, sgSmyd3-G600 ( i ), and sgShcbp1-G600 cells ( j ) in nude mice as shown by Western blots. k The cell growth curve of MDA-MB-231 (231) parental, sgSMYD3-231, and sgSHCBP1-231 cells was measured by IncuCyte. Tumor images of parental 231, sgSMYD3-231, and sgSHCBP1-231 cells ( l ) and plot of tumor weight ( m ) in nude mice (n = 12 mice/group). n The plot of relative spleen weight from the nude mice with the implantation of parental MDA-MB-231 (231), sgSMYD3-231, and sgSHCBP1-231 cells at 2 × 10 6 cells per mammary fat pad for 70 days (n = 12 mice/group). The protein levels of SMYD3, SHCBP1, KRAS, GRB2, pMEK, and pERK in tumors initiated with parental 231, sgSMYD3 ( o ), and sgSHCBP1 cells ( p ) in nude mice by Western blots. q Summary of tumor growth after disrupting either Smyd3 or Shcbp1 in mice.

Article Snippet: GRB2 (C-7) Antibody , Santa Cruz , sc-8034 , , 1:500.

Techniques: Control, Concentration Assay, MTT Assay, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: Representative tumor images ( a ), and tumor weight plots ( b ) at day 22 from FVB mice implanted with HP5712 cells at 1×10 6 cells per mammary fat pad, and treatment with PBS, PD1, trametinib (Tra), and PD1+Tra (n = 8 mice/group). c The plot of tumor volume in the processes of treatment in ( a , b ). d The plot of relative spleen weight from the same cohort of mice in a , b . Quantifications of PMN-MDSCs and M-MDSCs ( e ), CD4+ and CD8 + T cells ( f ) in CD45+ immune cell populations from the same cohorts of mice in a , b by CyTOF analysis at D12 (n = 3 mice/group). Quantifications of PMN-MDSCs and M-MDSCs ( g ), CD4+ and CD8 + T cells ( h ) in CD45+ immune cell populations from the same cohorts of mice in a , b by CyTOF analysis at D22 (n = 3 mice/group). i , j Tumor images from the HP Ctr mice, HP mice treated with αPD1 and Tra, and HP mice treated with αPD1 and Tra with depletion of T cell using CD8 antibody ( i ). The plot of tumor weight ( j ) in the same cohort of mice in ( i ) (n = 8 mice in each group). The protein levels of Smyd3 and Shcbp1 in tumors initiated with HP5712 cells and treatment with PBS, αPD1, Tra, and αPD1+Tra in FVB mice at D12 ( k ) and D22 ( l ) as shown by Western blots. The protein levels of pMek, pErk, Kras, and Grb2 in tumors initiated with HP5712 cells and treatment with PBS, αPD1, Tra, and αPD1+Tra in FVB mice at D12 ( m ) and D22 ( n ) as shown by Western blots. Expressions of Smyd3 ( o ) and Shcbp1 ( p ) in G600 cells with the treatment of E2, Tra, and E2 together with Tra at 1 hour, 2 hours, 4 hours, and 24 hours as determined by qPCR.

Article Snippet: GRB2 (C-7) Antibody , Santa Cruz , sc-8034 , , 1:500.

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: Primers used for ChIP-qPCR, qRT-PCR, and PCR.

Article Snippet: GRB2 (C-7) Antibody , Santa Cruz , sc-8034 , , 1:500.

Techniques:

Journal: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: Antibodies used for ChIP, IHC, IF, WB and in vivo mouse study.

Article Snippet: GRB2 (C-7) Antibody , Santa Cruz , sc-8034 , , 1:500.

Techniques: Immunohistochemistry-IF, In Vivo

Journal: Cell reports

Article Title: Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes

doi: 10.1016/j.celrep.2023.113603

Figure Lengend Snippet: A Mutations introduced to disrupt Sm-x-x-x-Sm dimerization motifs40 in the EGFR TM domain. The three Sm-x-x-x-Sm motifs are marked, where Sm is any small amino acid (red): T-g-m-v-G; G-m-v-g-A; and A-l-g-i-G. All Sm residues were replaced with valine in TM3X. B SPT data for wild type EGFR (gray) and a variant with the TM3X TM domain shown in A (blue). Unliganded receptors were tracked using QD-HA (open diamonds), and ligand-bound receptors were tracked using 200 pM QD-EGF (filled diamonds). EGF induces a similar slow-down for wild type EGFR (n = 40 cells without ligand, 42 with; P = 6.7 × 10−6) and the TM3X variant (n = 45 without ligand, 46 with; P = 1.9 × 10−7). Unpaired two-sided Welch’s t-tests were used to calculate P values (**P < 1×10−3; ***P < 1×10−6). See also Table S2. C pEGFR immunoblots of wild type and TM3X EGFR activated with 16 nM EGF for 5 min and probed with anti-pY1173 (upper), anti-EGFR (middle) and anti-GRB2 (lower) as loading control. Representative of three biological repeats.

Article Snippet: GRB2 antibody, polyclonal , Cell Signaling Technology , Cat#: 3972; RRID:AB_10693935.

Techniques: Variant Assay, Western Blot, Control

Journal: Cell reports

Article Title: Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes

doi: 10.1016/j.celrep.2023.113603

Figure Lengend Snippet: A SPT of EGFR with C-Tail phosphorylation sites removed by mutation or truncation. QD-EGF (200 pM) induced a significant slow-down for all variants compared with QD-HA-tracked unliganded EGFR, as shown for wild type EGFR (gray: P = 4.9 × 10−6; n = 124 cells without ligand, 56 with), Y9S (blood red: P = 1.8 × 10−4; n = 72 cells without ligand, 60 with), Y10X (medium red: P = 0.007; n = 59 without ligand, 68 with), and ΔC-Tail (red: P = 0.023; n = 166 cells without ligand, 118 with), but did reduce diffusion coefficient (ΔC-Tail + EGF compared with wild type + EGF; P = 1.9 ×10−3; n = 118 and 56 cells respectively). B Endogenous EGFR was tracked using QD-EGF (200 pM) in HeLa cells lacking GRB2 and SHC1 (GRB2/SHC1 KO: see Figure S6), and afatinib (10 μM) was added to reverse the slow-down (see Figure 5). The data suggest a significant EGF-induced slow-down in both wild type cells (gray, P = 6.7 × 10−21; n = 46 cells without afatinib, 46 with afatinib) and GRB2/SHC1 KO cells (orange, P = 1.6 × 10−9; n = 36 cells without afatinib, 42 with afatinib). Unpaired two-sided Welch’s t-tests were used to calculate P values (*P < 0.05; **P < 1×10−3; ***P < 1×10−6). See also Figures S5, S6, and Table S5.

Article Snippet: GRB2 antibody, polyclonal , Cell Signaling Technology , Cat#: 3972; RRID:AB_10693935.

Techniques: Phospho-proteomics, Mutagenesis, Diffusion-based Assay

Journal: Cell reports

Article Title: Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes

doi: 10.1016/j.celrep.2023.113603

Figure Lengend Snippet: Key resources table

Article Snippet: GRB2 antibody, polyclonal , Cell Signaling Technology , Cat#: 3972; RRID:AB_10693935.

Techniques: Polymer, Plasmid Preparation, Affinity Purification, Virus, Recombinant, Expressing, Software, Imaging

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 (RC200469), p85a-PI3K (RG210544) and SHIP2 (RC214716) vectors were obtained from Origene, CRKL (HG11261-CH) and STS1 (HG13868-NF) vectors were obtained from Sino Biological.

Techniques: Dissolution, Transfection, Western Blot, Sedimentation, Inhibition, Activity Assay, 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 (RC200469), p85a-PI3K (RG210544) and SHIP2 (RC214716) vectors were obtained from Origene, CRKL (HG11261-CH) and STS1 (HG13868-NF) vectors were obtained from Sino Biological.

Techniques: Lysis, SDS Page, Western Blot, Transfection, Mutagenesis, Inhibition, Activity 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: 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 (RC200469), p85a-PI3K (RG210544) and SHIP2 (RC214716) vectors were obtained from Origene, CRKL (HG11261-CH) and STS1 (HG13868-NF) vectors were obtained from Sino Biological.

Techniques: Transfection, Immunoprecipitation, Binding Assay, Western Blot, Plasmid Preparation, Activity 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: 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 (RC200469), p85a-PI3K (RG210544) and SHIP2 (RC214716) vectors were obtained from Origene, CRKL (HG11261-CH) and STS1 (HG13868-NF) vectors were obtained from Sino Biological.

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

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: ECM1 recruits GRB2 and SOS1 to the membrane to activate the MAPK signaling pathway. A) Affinity purification MS analysis of ECM1 interaction complexes in C4‐2B cells (left). Representative mass spectra of GRB2 and SOS1 peptides (right). B) IP analysis detected the interaction between ECM1 and GRB2 as well as SOS1 in C4‐2B cells with ECM1 (200 ng mL −1 ) treatment. C) IF staining and quantification of GRB2, SOS1, and DiI in C4‐2B cells treated with Veh (PBS) or ECM1 (200 ng mL −1 ). Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). D,E) WB analysis of GRB2 and SOS1 protein expression in whole lysis (WL) and membrane proteins from C4‐2B cells treated with increasing concentrations of ECM1 (0, 200, 400, 800 ng mL −1 ), or with the addition of either DMEM or CM. GAPDH was used as a loading control for whole lysis, and PMCA1 for membrane proteins. F) IP detection of the interaction between ECM1 and GRB2 as well as SOS1 in C4‐2B cells treated with CM. G) IF staining and quantification of GRB2, SOS1, and DiI in C4‐2B cells treated with DMEM or CM. Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). H,I) WB analysis of MEK, p‐MEK, ERK1/2, and p‐ERK1/2 expression in the indicated C4‐2B cells treated with or without ECM1 (200 ng mL −1 ). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Subsequently, cells were respectively incubated overnight at 4 °C with primary antibodies including ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:250), ENO1 Mouse mAb (Thermo Fisher Scientific; MA5‐47393, 1:2000), GRB2 Rabbit pAb (Proteintech; 10254‐2‐AP, 1:50) or SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:100).

Techniques: Membrane, Affinity Purification, Staining, Expressing, Lysis, Control

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Phosphorylated ENO1 bridges ECM1 with GRB2 and SOS1 at the membrane. A) Representative mass spectra of ENO1 peptide. B) IP analysis detected the interaction between ECM1 and ENO1 in C4‐2B cells. C) IF staining and quantification of ENO1 and ECM1 in C4‐2B cells. Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). D) Proximity ligation assay (PLA) analysis of the interaction between ECM1‐Flag and ENO1 (Scale bar, 10 µm). E) IP assays of the interaction between ENO1 and GRB2 as well as SOS1 in C4‐2B cells in the presence or absence of ECM1 (200 ng mL −1 ). F) IP analysis detected the interaction between ECM1 and GRB2 as well as SOS1 in the indicated C4‐2B cells treated with ECM1 (200 ng mL −1 ). G) Expression of GRB2 and SOS1 protein in whole lysis and membrane proteins from the indicated C4‐2B cells with ECM1 (200 ng mL −1 ) treatment by WB analysis. H) IF staining and quantification of GRB2, SOS1, and DiI in the indicated C4‐2B cells supplemented with ECM1 (200 ng mL −1 ). Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). I) Representative peptides showing phosphorylation of ENO1 at the Y189 site by affinity purification MS analysis of protein modifications in ECM1‐treated C4‐2B cells. J) Tyr phosphorylation detection of the immunoprecipitated ENO1 in indicated C4‐2B and LNCap cells with ECM1 treatment (200 ng mL −1 ). K) Tyr phosphorylation levels of HA‐ENO1‐WT and ‐Y189F in C4‐2B cells with or without ECM1 (200 ng mL −1 ) treatment. L) IF staining and quantification of GRB2, SOS1, and DiI in the indicated C4‐2B cells supplemented with ECM1 (200 ng mL −1 ). Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). M,N) IP assays of the interaction between ECM1 and ENO1, GRB2 as well as SOS1 in the indicated C4‐2B cells in the presence of ECM1 (200 ng mL −1 ). O) WB analysis of MEK, p‐MEK, ERK1/2, and p‐ERK1/2 expression in the indicated C4‐2B cells treated with or without ECM1 (200 ng mL −1 ). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Subsequently, cells were respectively incubated overnight at 4 °C with primary antibodies including ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:250), ENO1 Mouse mAb (Thermo Fisher Scientific; MA5‐47393, 1:2000), GRB2 Rabbit pAb (Proteintech; 10254‐2‐AP, 1:50) or SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:100).

Techniques: Membrane, Staining, Proximity Ligation Assay, Expressing, Lysis, Phospho-proteomics, Affinity Purification, Immunoprecipitation

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: PhAH attenuates ENO1‐mediated PCa cell resistance to ENZ. A) Chemical structure of PhAH. B) Ligand interaction diagram (left) and binding amino acid residue sites (right) of the highest‐scoring PhAH and ENO1 protein molecular docking complex (RMSD = 1.9977; E_score = −3.9538). C) WB detection of the interaction between ECM1 and ENO1, as well as Tyr phosphorylation of ENO1 following immunoprecipitating ENO1 in C4‐2B cells in the presence of ECM1 (200 ng mL −1 ) with or without PhAH (1 µM). D) IF staining and quantification of GRB2, SOS1, and DiI in C4‐2B cells treated with or without ECM1 (200 ng mL −1 ) or PhAH (1 µM). Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). E) Representative images (top) and quantification (bottom) of surviving colonies formed by C4‐2B and LNCaP cells treated with or without ECM1 (200 ng mL −1 ) or PhAH (1 µM). F) Flow cytometry analysis showing representative images (top) and quantification (bottom) of apoptosis in C4‐2B and LNCaP cells with or without ECM1 (200 ng mL −1 ) or PhAH (1 µM) treatment. G) Representative BLI of intratibial tumors in mice treated daily oral treatment with Veh, ENZ (20 mg kg −1 ), ENZ (20 mg kg −1 ) combined with intraperitoneal (i.p.) injection of either Veh (DMSO) or PhAH (5 mg kg −1 ) twice weekly at 4 weeks before treatment, and at weeks 0, 6, and 8 during treatment (n = 6 per group). H) Quantification of BLI signals in intratibial tumors of mice before and after Tx as grouped in G (n = 6 per group). I) Representative micro‐CT images of intratibial lesions from mice after 8 weeks of treatment grouped as shown in G (arrows and circles indicate osteoblastic lesions, n = 6 per group). J) Representative H&E images of intratibial tumors grouped as shown in G (T, tumor; N, the adjacent non‐tumor tissues. Scale bars, 500 µm and 100 µm). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Subsequently, cells were respectively incubated overnight at 4 °C with primary antibodies including ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:250), ENO1 Mouse mAb (Thermo Fisher Scientific; MA5‐47393, 1:2000), GRB2 Rabbit pAb (Proteintech; 10254‐2‐AP, 1:50) or SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:100).

Techniques: Binding Assay, Residue, Phospho-proteomics, Staining, Flow Cytometry, Injection, Micro-CT

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Schematic diagram illustrating that increased osteoblast‐derived ECM1 from the bone microenvironment of BMPC patients induced by ENZ treatment, interacts with the ENO1 receptor on the prostate cancer cell membrane, further recruiting adapter proteins including GRB2 and SOS1, which activates the downstream MAPK signaling pathway to promote the proliferation of PCa cells and induce anti‐androgen resistance.

Article Snippet: Subsequently, cells were respectively incubated overnight at 4 °C with primary antibodies including ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:250), ENO1 Mouse mAb (Thermo Fisher Scientific; MA5‐47393, 1:2000), GRB2 Rabbit pAb (Proteintech; 10254‐2‐AP, 1:50) or SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:100).

Techniques: Derivative Assay, Membrane

Journal: Cell Reports Medicine

Article Title: A RET::GRB2 fusion in pheochromocytoma defies the classic paradigm of RET oncogenic fusions

doi: 10.1016/j.xcrm.2022.100686

Figure Lengend Snippet: RET::GRB2 fusion is detected as a somatic event in a pheochromocytoma (A) Representation of the region spanning the RET exon 18 (left panel) and GRB2 exon 3 (right panel) in a pheochromocytoma predicted to carry the RET::GRB2 fusion, indicating the number of spanning reads (n = 18) and junction reads (n = 492) detected by RNA-seq. Chromosome (chr) location is indicated for each gene. (B) Chromosomes (chrs) plotted as ideograms around the outside of the circle (CIRCOS) plot depicting the putative recombination between RET (chr10) and GRB2 (chr 17). The thickness of the connecting line represents the depth of read coverage. (C) Hematoxylin and eosin (H&E) staining of the primary composite tumor comprised of a predominant pattern of pheochromocytoma (right) combined with a focal area of ganglioneuroma (left); magnification 100×, scale bar, 100 μm (inset, 400×, scale bar, 20 μm). (D) Agarose gel of PCR products spanning the RET::GRB2 fusion transcript in tumor but not matched blood (leukocyte) cDNA; two other paired blood/tumor cDNA samples from patients with a RET mutation and a SDHB germline mutation, respectively, show no product (top); housekeeping gene TBP is shown (bottom). These results were repeated three times. (E) Sanger sequencing of the product shown in (D), displaying RET sequence (exon 18) merged with GRB2 (exon 3). This experiment was repeated three times. (F) RET (left) and GRB2 (right) mRNA expression obtained from high-throughput sequencing (HT-seq) counts of RNA-seq from 30 pheochromocytomas/paragangliomas (PPGLs; dots), depicting an exon within the fusion (x axis) and one outside the fusion (y axis). RET::GRB2- positive tumor is indicated in blue. (G) DNA-based quantitative real-time PCR assay for copy-number assessment of the RET and GRB2 genes using primers targeting the area within (x axis) or outside the fusion (y axis) of a set of 14 PPGLs of various genotypes (dots). This experiment was repeated three times in duplicate samples.

Article Snippet: Membranes were blocked in 5% skim milk and then probed at 4°C overnight with the following primary antibodies: phosphorylated RET-Y905 [Cell Signaling Technology (CST) 3221, 1:750 dilution], phosphorylated RET-Y1062 (Abcam ab51103, 1:750), RET E1N8X (CST 14556, 1:1500), RET (Abcam ab134100, 1:1200), phosphorylated AKT-S473 (CST 9271, 1:750), AKT (CST 9272, 1:1000), phosphorylated ERK T202/Y204(CST 4377, 1:1500), ERK1/ERK2 (CST 4695, 1:1500), GRB2 (CST 36344, 1:2000), PARP (CST 9542, 1:1000), β-actin (CST 3700, 1:2000), α-tubulin (CST 3873, 1:2000).

Techniques: RNA Sequencing, Staining, Agarose Gel Electrophoresis, Mutagenesis, Sequencing, Expressing, Next-Generation Sequencing, Real-time Polymerase Chain Reaction

Journal: Cell Reports Medicine

Article Title: A RET::GRB2 fusion in pheochromocytoma defies the classic paradigm of RET oncogenic fusions

doi: 10.1016/j.xcrm.2022.100686

Figure Lengend Snippet: Validation of the RET::GRB2 fusion protein in the pheochromocytoma and in vitro (A) Diagram of wild-type (WT) RET, displaying relevant domains as indicated (CadLD, cadherin-like domains; CRD, cysteine-rich domain; TM, transmembrane domain; TKD, tyrosine kinase domain). The two main RET isoforms diverge at amino acid (aa) 1,063 (∗) with either 51 (RET51) or 9 (RET9) distinctive aa at the C terminus; WT GRB2 contains two SH3 domains flanking one SH2 domain. The RET (R1013) and GRB2 (V27) breakpoint sites are indicated; full-length RET::GRB2 fusion spans 1,204 aa. (B) Western blot of protein lysates from PPGLs carrying mutations in RET , SDHB , VHL , and EPAS1 genes, and the RET::GRB2 fusion, probed with phosphorylated (P) RET (Y905) and two distinct total (T) RET antibodies directed at the extracellular, N-terminal (N-term) region around D320 and C-terminal (C-term) region beyond aa 1,100. GRB2 shows both the WT and fusion product, P- and T- AKT; ERK1/2, β-actin is a loading control; two technical replicates were performed. (C) SH-SY5Y cells stably expressing RET9, RET51, RET::GRB2, and a control vector, probed with RET, GRB2, and α-tubulin as a loading control; three biological replicates were performed. (D) SH-SY5Y cells expressing RET::GRB2 or RET51 were starved of serum for 3 h and exposed to 100 ng/mL GDNF (+) or vehicle (−) for 10 min, and lysates were probed with P-RET Y905 or T-RET; α-tubulin is a loading control; experiments were repeated three times. (E) SH-SY5Y cells expressing RET::GRB2, RET 9, or RET51 constructs were treated with GDNF as in (D). Lysates were probed with GRB2 and T- and P- AKT, ERK1/2, T RET, and RET phosphorylated at Y1062, a region excluded from the RET::GRB2 fusion; α-tubulin is a loading control; experiments were repeated three times. (F) Confocal microscopy of HEK293T cells expressing WT, mutant (C634R) RET, or RET::GRB2 fusion, labeled with a tag antibody in green (MYC for WT and C634R RET or hemagglutinin [HA] for RET::GRB2 fusion) and a membrane marker, Na/K ATPase (red). Nuclei are stained with DAPI (blue). A merged image is shown in the right panels. Scale bar: 10 μm. (G) Quantification of the colocalized signals between RET and the Na/K ATPase using ImageJ from multiple independent images (n = 10–13 cells/genotype, two biological replicates). One way ANOVA, p = 0.048; ∗p = 0.02, two-tailed t test; ∗∗p = 0.0042, two-tailed t test. (H) Uniform manifold approximation and projection (UMAP) plot of RNA-seq data from pheochromocytomas/PPGLs of our cohort (n = 30, UTHSA) and TCGA (n = 178), color-coded by genotype; gray symbols are tumors with unknown mutations; RET::GRB2 fusion (red triangle).

Article Snippet: Membranes were blocked in 5% skim milk and then probed at 4°C overnight with the following primary antibodies: phosphorylated RET-Y905 [Cell Signaling Technology (CST) 3221, 1:750 dilution], phosphorylated RET-Y1062 (Abcam ab51103, 1:750), RET E1N8X (CST 14556, 1:1500), RET (Abcam ab134100, 1:1200), phosphorylated AKT-S473 (CST 9271, 1:750), AKT (CST 9272, 1:1000), phosphorylated ERK T202/Y204(CST 4377, 1:1500), ERK1/ERK2 (CST 4695, 1:1500), GRB2 (CST 36344, 1:2000), PARP (CST 9542, 1:1000), β-actin (CST 3700, 1:2000), α-tubulin (CST 3873, 1:2000).

Techniques: Biomarker Discovery, In Vitro, Western Blot, Control, Stable Transfection, Expressing, Plasmid Preparation, Construct, Confocal Microscopy, Mutagenesis, Labeling, Membrane, Marker, Staining, Two Tailed Test, RNA Sequencing

Journal: Cell Reports Medicine

Article Title: A RET::GRB2 fusion in pheochromocytoma defies the classic paradigm of RET oncogenic fusions

doi: 10.1016/j.xcrm.2022.100686

Figure Lengend Snippet: RET::GRB2 fusion is oncogenic and sensitive to clinical grade RET inhibitors (A) Constructs used to evaluate transforming activity in Ba/F3 cells: (domains are depicted as in A); RET-C634R, pathogenic RET mutant; RET truncated, first 1,013 amino acids of RET, containing only the RET component of the fusion; RET::GRB2, contains the RET::GRB2 fusion; RET::GRB2-KD, contains the kinase-dead version of the RET::GRB2 fusion carrying a K758 M mutation in the RET catalytic domain. (B) Western blot of lysates from Ba/F3 cells stably expressing the constructs indicated in (A), along with parental cells, probed for total RET, and α-tubulin as a loading control; these experiments were repeated 3 times. (C) Growth rate of Ba/F3 cells stably expressing the constructs indicated in (A) and (B), and parental cells cultured in the absence of interleukin 3 (IL-3). Cells were plated in triplicate and counted daily for 4 days; experiments were repeated 3 times. ∗∗∗∗p < 0.0001, two-way ANOVA. (D) IC50 concentration-response curves to selpercatinib at doses of 0, 6.25, 12.5, 25, 50, 100, and 400 nM for 72 h measuring inhibition of growth of Ba/F3 cells expressing the RET::GRB2 fusion (4.1 nM, 95% CI, 3.4–4.9 nM) or RET-C634R mutant (29.9 nM, 95% CI, 21.2–42.6 nM) seeded in triplicate per dose and repeated three times. (E) Lysates from Ba/F3 cells expressing RET::GRB2 or RET-C634R treated with 25 or 100 nM selpercatinib or vehicle for 4 h were probed with P-RET (Y905), and α-tubulin as loading control; three biological replicates were performed. s.e. and l.e. indicate short and long immunoblot exposure, respectively.

Article Snippet: Membranes were blocked in 5% skim milk and then probed at 4°C overnight with the following primary antibodies: phosphorylated RET-Y905 [Cell Signaling Technology (CST) 3221, 1:750 dilution], phosphorylated RET-Y1062 (Abcam ab51103, 1:750), RET E1N8X (CST 14556, 1:1500), RET (Abcam ab134100, 1:1200), phosphorylated AKT-S473 (CST 9271, 1:750), AKT (CST 9272, 1:1000), phosphorylated ERK T202/Y204(CST 4377, 1:1500), ERK1/ERK2 (CST 4695, 1:1500), GRB2 (CST 36344, 1:2000), PARP (CST 9542, 1:1000), β-actin (CST 3700, 1:2000), α-tubulin (CST 3873, 1:2000).

Techniques: Construct, Activity Assay, Mutagenesis, Western Blot, Stable Transfection, Expressing, Control, Cell Culture, Concentration Assay, Inhibition

Journal: Cell Reports Medicine

Article Title: A RET::GRB2 fusion in pheochromocytoma defies the classic paradigm of RET oncogenic fusions

doi: 10.1016/j.xcrm.2022.100686

Figure Lengend Snippet:

Article Snippet: Membranes were blocked in 5% skim milk and then probed at 4°C overnight with the following primary antibodies: phosphorylated RET-Y905 [Cell Signaling Technology (CST) 3221, 1:750 dilution], phosphorylated RET-Y1062 (Abcam ab51103, 1:750), RET E1N8X (CST 14556, 1:1500), RET (Abcam ab134100, 1:1200), phosphorylated AKT-S473 (CST 9271, 1:750), AKT (CST 9272, 1:1000), phosphorylated ERK T202/Y204(CST 4377, 1:1500), ERK1/ERK2 (CST 4695, 1:1500), GRB2 (CST 36344, 1:2000), PARP (CST 9542, 1:1000), β-actin (CST 3700, 1:2000), α-tubulin (CST 3873, 1:2000).

Techniques: Purification, Virus, Recombinant, Reverse Transcription, SYBR Green Assay, Sequencing, Software

Journal: bioRxiv

Article Title: Quantitation of FGFR3 signaling via GRB2 recruitment on micropatterned surfaces

doi: 10.1101/2022.04.11.487861

Figure Lengend Snippet: (a , b) Antibody patterns are used to enrich and immobilize mGFP-FGFR3 at specific sites (“ON” regions) in the plasma membrane of HeLa cells, leaving other regions depleted of mGFP-FGFR3 (“OFF”). Co-localization of the adaptor protein GRB2-mScarlet to mGFP-FGFR3 patterns reports on the activation state of FGFR3, with no or little co-patterning observable in the non-activated state (a) and a high degree of co-patterning for the activated receptor after addition of the ligand fgf1 (b). TIR illumination is used to specifically detect membrane-proximal protein. (c) The fluorescence contrast of GRB2-mScarlet (C mScarlet ) relates the fluorescence intensity within ON (I ON,mGFP ) and OFF (I OFF,mGFP ) areas of FGFR3-enriched regions and serves to quantify the extent of co-localization. Each dot represents one cell. C mScarlet data for the wildtype receptor (WT), a kinase-dead mutant (K508M) and a mGFP-FGFR3-mScarlet fusion protein as positive control is shown (p-value annotation legend: *= 0.01 ≤ p ≤ 0.05; **= 0.001 ≤ p ≤ 0.01; ***= 0.0001 ≤ p ≤ 0.001; ****= p ≤ 0.0001). (d , e) Correlation between the receptor’s intensity in ON (d) and OFF (e) regions and the GRB2-mScarlet contrast for the WT receptor. Data in the absence (black) and presence (orange) of fgf1 is shown. The grey box indicates the cell population with C mScarlet < 0.2, which likely represents non-activated cells. (f , g) Correlation between GRB2-mScarlet contrast and mGFP-FGFR3 intensity in ON (f) and OFF (g) regions for K508M. All correlation coefficients can be found in Supplementary Table S1

Article Snippet: To obtain the GRB2-mScarlet plasmid, we carried out PCR to amplify the mScarlet sequence from the ITPKA-mScarlet plasmid (Addgene, USA) as well as the GRB2 sequence from GRB2-YFP (gift from J. Weghuber, FH Wels, Austria) with >15 nt overhangs complementary to adjacent regions on the target plasmid.

Techniques: Clinical Proteomics, Membrane, Activation Assay, Fluorescence, Mutagenesis, Positive Control

Journal: International journal of molecular medicine

Article Title: Gab2 promotes the growth of colorectal cancer by regulating the M2 polarization of tumor‑associated macrophages.

doi: 10.3892/ijmm.2023.5327

Figure Lengend Snippet: Figure 1. Gab2 is upregulated within TAMs in tumor tissues and is associated with the poor prognosis of patients with CRC. (A) Expression level of Gab2 in CRC and adjacent normal tissues from The Cancer Genome Atlas database. ****P<0.0001, tumor vs. normal. (B) Representative images of immunohistochem‑ ical staining of Gab2 and CD68 in colorectal carcinoma and para‑cancerous tissues. (C) Multiplex immunofluorescence staining of the macrophage markers, CD68 and Gab2. CD68 staining is shown in green, Gab2 is shown in red, and DAPI staining in blue. The panels on the right of each image are enlarged images of the boxed area in the main images. (D) The association between Gab2 expression in TAMs and the 5‑year survival rate of patients with CRC. According to the median of the immunofluorescence intensity score, the patients with CRC were divided into two groups (Gab2 low expression and Gab2 high expression). Survival curves were plotted using the Kaplan‑Meier method, and the statistical significance of the difference in 5‑year survival rates between the groups was assessed using the log‑rank test. *P<0.05. Gab2, Gab2, Grb2‑associated binder 2; TAMs, tumor‑associated macrophages; CRC, colorectal cancer.

Article Snippet: The following antibodies were used in the present study: CD68 (1:100; cat. no. ab283654; Abcam), Gab2 (1:50; cat. no. 22549‐1‐AP; Proteintech Group, Inc.).

Techniques: Expressing, Staining, Multiplex Assay, Immunofluorescence

Journal: International journal of molecular medicine

Article Title: Gab2 promotes the growth of colorectal cancer by regulating the M2 polarization of tumor‑associated macrophages.

doi: 10.3892/ijmm.2023.5327

Figure Lengend Snippet: Figure 2. TAMs in tumor tissues exhibit a higher expression of Gab2 and the M2 phenotype. (A) Diagram of the simulated tumor microenvironment in vitro. (B‑D) Gab2 expression levels in TCM‑stimulated PMΦ. (E and F) Gab2 expression levels in TCM‑stimulated BMDM. (G) Gab2 expression in TCM‑stimulated THP‑1 cells. (H) The expression of Gab2 in TAMs harvested from CT26 tumor‑bearing mice and the percentage of macrophages was analyzed using fluo‑ rescence‑activated cell sorting. (I) The Gab2 mRNA expression levels in Tu‑TAM were measured using reverse transcription‑quantitative PCR. (J) The protein expression of Gab2 in Tu‑TAM was detected using western blot analysis. **P<0.01, Tu‑TAM vs. PMΦ. TAMs, tumor‑associated macrophages; Gab2, Gab2, Grb2‑associated binder 2; TCM, tumor‑conditioned medium; PMΦ, peritoneal macrophages; BMDM, bone marrow‑derived macrophages; Tu‑TAM, macrophages sorted from subcutaneously transplanted tumors in mice.

Article Snippet: The following antibodies were used in the present study: CD68 (1:100; cat. no. ab283654; Abcam), Gab2 (1:50; cat. no. 22549‐1‐AP; Proteintech Group, Inc.).

Techniques: Expressing, In Vitro, FACS, Western Blot

Journal: International journal of molecular medicine

Article Title: Gab2 promotes the growth of colorectal cancer by regulating the M2 polarization of tumor‑associated macrophages.

doi: 10.3892/ijmm.2023.5327

Figure Lengend Snippet: Figure 3. Expression of TAM polarization‑related molecules. PMΦ from BALB/c mice were stimulated with LPS + IFN‑γ and IL‑4 for 24 h, serving as an M1/M2 positive control. (A) Evaluation of Inos, Il‑12 and Cxcl9 mRNA expression levels in PMΦ, Tu‑TAM, TCM‑TAM using RT‑qPCR. **P<0.01, Tu‑TAM, TCM‑TAM, IL‑4, LPS + IFN‑γ vs. PMΦ. (B) Evaluation of Il‑10, Arg‑1, Ym‑1, Fizz1, Ccl17, Vegf mRNA expression levels in PMΦ, Tu‑TAM, TCM‑TAM using RT‑qPCR. *P<0.05, Tu‑TAM, TCM‑TAM vs. PMΦ. **P<0.01, Tu‑TAM, TCM‑TAM, IL‑4 vs. PMΦ. (C) Evaluation of Arg‑1, CD206 protein levels in PMΦ, Tu‑TAM, TCM‑TAM using western blot analysis. **P<0.01, Tu‑TAM, TCM‑TAM, IL‑4 vs. PMΦ. TAMs, tumor‑associated macrophages; PMΦ, peritoneal macrophages; LPS, LPS, lipopolysaccharide; IFN‑γ, interferon‑γ; IL, interleukin; Inos, inducible nitric oxide synthase; Cxcl9, C‑X‑C motif chemokine ligand 9; Tu‑TAM, macrophages sorted from subcutaneously transplanted tumors in mice; TCM, tumor‑conditioned medium; RT‑qPCR, reverse transcription‑quantitative PCR; Arg‑1, arginase‑1; Ym‑1, Chil3/chitinase‑like protein 3; Fizz1, Retnla/resistin‑like molecule alpha; Vegf, vascular endothelial growth factor; Ccl17, C‑C motif chemokine ligand 17; Gab2, Gab2, Grb2‑associated binder 2.

Article Snippet: The following antibodies were used in the present study: CD68 (1:100; cat. no. ab283654; Abcam), Gab2 (1:50; cat. no. 22549‐1‐AP; Proteintech Group, Inc.).

Techniques: Expressing, Positive Control, Western Blot

Journal: International journal of molecular medicine

Article Title: Gab2 promotes the growth of colorectal cancer by regulating the M2 polarization of tumor‑associated macrophages.

doi: 10.3892/ijmm.2023.5327

Figure Lengend Snippet: Figure 4. Suppression of Gab2 expression impedes the M2 polarization of TAMs. (A) Visualization of EGFP expression in PMΦ following lentivirus infec‑ tion. Scale bar, 50 µm. (B and C) The expression of Gab2 in PMΦ post‑lentivirus infection. Scar bar, 10 µm. **P<0.01, LV‑Gab2 vs. respective control (D) Effect of the suppression of Gab2 expression on the molecules related to TAM M1 polarization. *P<0.05, TCM‑LV‑Con vs. LV‑Con. **P<0.01, LV‑Gab2 vs. LV‑Con. †P<0.05, TCM‑LV‑Gab2 vs. TCM‑LV‑Con. (E) Effect of the suppression of Gab2 expression on TAM M2 polarization markers. *P<0.05, LV‑Gab2 vs. LV‑Con. **P<0.01, TCM‑LV‑Con, LV‑Gab2 vs. PMΦ. †P<0.05, TCM‑LV‑Gab2 vs. TCM‑LV‑Con. (F) Effect of the suppression of Gab2 expression on TAM M2 polarization markers. *P<0.05, TCM‑LV‑Con, LV‑Gab2 vs. LV‑Con. **P<0.01, TCM‑LV‑Con vs. PMΦ. †P<0.05, TCM‑LV‑Gab2 vs. LV‑Con. Gab2, Gab2, Grb2‑associated binder 2; TAMs, tumor‑associated macrophages; PMΦ, peritoneal macrophages; IL, interleukin; Arg‑1, arginase‑1; Ym‑1, Chil3/chitinase‑like protein 3; Fizz1, Retnla/resistin‑like molecule alpha; Vegf, vascular endothelial growth factor; Ccl17, C‑C motif chemokine ligand 17.

Article Snippet: The following antibodies were used in the present study: CD68 (1:100; cat. no. ab283654; Abcam), Gab2 (1:50; cat. no. 22549‐1‐AP; Proteintech Group, Inc.).

Techniques: Expressing, Infection, Control

Journal: International journal of molecular medicine

Article Title: Gab2 promotes the growth of colorectal cancer by regulating the M2 polarization of tumor‑associated macrophages.

doi: 10.3892/ijmm.2023.5327

Figure Lengend Snippet: Figure 5. Suppression of Gab2 expression attenuates TAM‑mediated CRC tumorigenesis. (A) Diagram of subcutaneous xenograft model. (B) Tumorigenesis assay of BALB/c mice subcutaneously injected with CT26 cells (n=3), Gab2WT‑CT26 cells (n=3) and Gab2Def‑CT26 cells (n=3). (C) Tumor growth curve of the CT26, Gab2WT‑CT26 and Gab2Def‑CT26 groups. (D) Representative photos of harvested tumors from the different experimental groups (left). Scar bar, 15 mm, and the corresponding tumor weight (right). *P<0.05, Gab2WT‑CT26 group vs. CT26 group; †P<0.05, Gab2Def‑CT26 group vs. Gab2WT‑CT26 group. (E) Histopathological analysis of tumor and lung tissues visualized using hematoxylin and eosin staining. (F) Immunofluorescence was performed to detect the expression of Gab2 and M2 polarization markers CD206 and Arg‑1 in tumor tissue TAMs. The panels on the right of each image are enlarged images of the boxed area in the main images. Gab2, Grb2‑associated binder 2; TAMs, tumor‑associated macrophages; Arg‑1, arginase‑1.

Article Snippet: The following antibodies were used in the present study: CD68 (1:100; cat. no. ab283654; Abcam), Gab2 (1:50; cat. no. 22549‐1‐AP; Proteintech Group, Inc.).

Techniques: Expressing, Injection, Staining, Immunofluorescence

Journal: International journal of molecular medicine

Article Title: Gab2 promotes the growth of colorectal cancer by regulating the M2 polarization of tumor‑associated macrophages.

doi: 10.3892/ijmm.2023.5327

Figure Lengend Snippet: Figure 6. Gab2 induces M2‑like macrophage polarization through the AKT/ERK signaling pathway. (A) The expression levels of p‑AKT, p‑ERK, p‑STAT6 and p‑STAT3 were measured using western blot analysis. (B) Quantitative evaluation of the expression levels of p‑AKT, p‑ERK, p‑STAT6, p‑STAT3. **P<0.01, TCM‑LV‑Con vs. LV‑Con. †P<0.05, TCM‑LV‑Gab2 vs. TCM‑LV‑Con. Grb2‑associated binder 2; TCM, tumor‑conditioned medium; STAT, signal transducer and activator of transcription.

Article Snippet: The following antibodies were used in the present study: CD68 (1:100; cat. no. ab283654; Abcam), Gab2 (1:50; cat. no. 22549‐1‐AP; Proteintech Group, Inc.).

Techniques: Expressing, Western Blot