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: BMC Cancer

Article Title: Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization

doi: 10.1186/s12885-015-1415-6

Figure Lengend Snippet: Evaluation of compounds for their effect on internalization of Grb2. a Schematic of EGFR internalization cycle through Grb2-positive early endosomes ( EE ) and late endosomes ( LE ) . b SCC61 cells treated with vehicle or EGF, visualized with antibody to Grb2. Right panels represent image overlayed with object mask generated by the Transfluor module. c Representative fields for untreated versus EGF-treated cells pre-incubated with controls (vehicle, erlotinib, cetuximab) or indicated compounds. d Quantitation of changes in pit count ratio; numbers over the bars indicate the docking boxes for each compound; + indicates that docking pose predicts EGF interaction. Class 1 and 2 compounds are reported comparing EGF-to-vehicle ratio in the absence of compounds versus EGF-to-vehicle ratio in the presence of compound, representative screening data. Compound classes are indicated. Representative screening data for Class 4 compounds are reported comparing pit count ratio for compound alone normalized to vehicle indicating activity in the absence of EGF

Article Snippet: Cells were incubated with primary Grb2 antibody (#3972, Cell Signaling, Danvers, MA used at 1:500 in 0.2 % BSA) for 2 h at room temperature.

Techniques: Generated, Incubation, Quantitation Assay, Activity Assay

Journal: Scientific Reports

Article Title: iTRAQ-based Proteomic Analysis of Porcine Kidney Epithelial PK15 cells Infected with Pseudorabies virus

doi: 10.1038/srep45922

Figure Lengend Snippet: Immunoblotting analysis of proteins (beta-catenin, STAT1, GRB2 and PCNA) in PRV-infected or mock-infected PK15 cells. WB ratios and iTRAQ ratios (infection/mock) were shown on the right side. The β-actin protein was used as a control.

Article Snippet: The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and then incubated overnight at 4 °C with primary antibodies specific for β-Actin (4967, Cell Signaling Technology), beta-catenin (51067-2-AP), STAT1 (10144-2-AP), GRB2 (10254-2-AP) and PCNA (60097-1-Ig) purchased from Proteintech Group.

Techniques: Western Blot, Infection, Multiplex sample analysis, 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

Journal: Frontiers in Immunology

Article Title: Identification of tumor associated neutrophils-related genes in triple-negative breast cancer for predicting prognosis and therapeutic response through integrated single-cell analysis

doi: 10.3389/fimmu.2025.1613529

Figure Lengend Snippet: Identification of candidate genes. (A) Differences in survival between the high and low neutrophil group. (B) RSF analysis and expression levels of genes related to prognosis. (C) Expression levels and distributions of RASGRP4, COX20, CD47, TIMM10B, LY86, GRAP, TNFRSF13C and ATP6V0D1 in different tumor subtypes. (D-G) Kaplan–Meier graphs displaying the survival potential of patients with TNBC, grouped by the expression levels of significant genes.

Article Snippet: The primary antibodies used in the experiment included antibodies against MPO (ZSGB-Bio, ZA-0197, diluted1:200), TIMM10B (Proteintech, 10907-1, diluted 1:200), GRAP (Proteintech, 14505-1, diluted 1:200), and TNFRSF13C (Proteintech, 22582-1, diluted 1:200), RASGRP4 (Abcam, 96293, diluted 1:100).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Identification of tumor associated neutrophils-related genes in triple-negative breast cancer for predicting prognosis and therapeutic response through integrated single-cell analysis

doi: 10.3389/fimmu.2025.1613529

Figure Lengend Snippet: Correlations between TIMM10B, GRAP, TNFRSF13C and RASGRP4 and drug sensitivity. (A-D) Correlations between key genes and the IC50 of chemotherapeutic agents.

Article Snippet: The primary antibodies used in the experiment included antibodies against MPO (ZSGB-Bio, ZA-0197, diluted1:200), TIMM10B (Proteintech, 10907-1, diluted 1:200), GRAP (Proteintech, 14505-1, diluted 1:200), and TNFRSF13C (Proteintech, 22582-1, diluted 1:200), RASGRP4 (Abcam, 96293, diluted 1:100).

Techniques:

Journal: Frontiers in Immunology

Article Title: Identification of tumor associated neutrophils-related genes in triple-negative breast cancer for predicting prognosis and therapeutic response through integrated single-cell analysis

doi: 10.3389/fimmu.2025.1613529

Figure Lengend Snippet: GSEA and GSVA of the four genes. (A-D) . GSEA revealed the enriched signaling pathways associated with TIMM10B, GRAP, TNFRSF13C and RASGRP4. (E-H) . Analysis of key genes using GSVA. The x-axis illustrates the t value of the GSVA score, and the y-axis depicts KEGG pathways; blue highlights upregulated pathways, whereas green highlights downregulated pathways. |NES| ≥ 1 and FDR < 0.25.

Article Snippet: The primary antibodies used in the experiment included antibodies against MPO (ZSGB-Bio, ZA-0197, diluted1:200), TIMM10B (Proteintech, 10907-1, diluted 1:200), GRAP (Proteintech, 14505-1, diluted 1:200), and TNFRSF13C (Proteintech, 22582-1, diluted 1:200), RASGRP4 (Abcam, 96293, diluted 1:100).

Techniques: Protein-Protein interactions

Journal: Frontiers in Immunology

Article Title: Identification of tumor associated neutrophils-related genes in triple-negative breast cancer for predicting prognosis and therapeutic response through integrated single-cell analysis

doi: 10.3389/fimmu.2025.1613529

Figure Lengend Snippet: Cell communication and quasitemporal analysis. (A) Circus plot illustrating the greater total number of significantly interacting pairs between neutrophils and immune cells as estimated by CellPhoneDB (P<0.05). (B) Bubble diagram of the cell communication network between ligands and neutrophils and other cell subtypes as well as with neutrophil itself themselves. (C-E) Trajectory analysis of the potential relatedness between the two groups according to pseudotime, cell type and group. (F-H) Changes in the expression of TIMM10B, GRAP, TNFRSF13C and RASGRP4 over pseudotime.

Article Snippet: The primary antibodies used in the experiment included antibodies against MPO (ZSGB-Bio, ZA-0197, diluted1:200), TIMM10B (Proteintech, 10907-1, diluted 1:200), GRAP (Proteintech, 14505-1, diluted 1:200), and TNFRSF13C (Proteintech, 22582-1, diluted 1:200), RASGRP4 (Abcam, 96293, diluted 1:100).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Identification of tumor associated neutrophils-related genes in triple-negative breast cancer for predicting prognosis and therapeutic response through integrated single-cell analysis

doi: 10.3389/fimmu.2025.1613529

Figure Lengend Snippet: Clinical relevance of TIMM10B, GRAP, TNFRSF13C and RASGRP4. (A) Polychromatic immunofluorescence staining showing the distribution of MPO, TIMM10B, GRAP, TNFRSF13C and RASGRP4 expression. Scale bar (upper panel), 200 µm. Scale bar (bottom panel), 50 µm. (B) IHC scores of MPO, TIMM10B, GRAP, TNFRSF13C and RASGRP4 in adjacent tissue (AT), stage I-II (I-II) and III TNBC. (C) Correlations between MPO and the four candidate genes in stage I-II (I-II) TNBC. (D) Correlations between MPO and the four candidate genes in stage III (III) TNBC. (E) Representative images of coimmunostaining for MPO and four target genes in adjacent tissue from the stage I-II and III TNBC groups. Scale bar, 20 µm. (F) Percentage of TIMM10B-, GRAP-, TNFRSF13C- and RASGRP4-positive cells in AT and stage I-II and III TNBC. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Article Snippet: The primary antibodies used in the experiment included antibodies against MPO (ZSGB-Bio, ZA-0197, diluted1:200), TIMM10B (Proteintech, 10907-1, diluted 1:200), GRAP (Proteintech, 14505-1, diluted 1:200), and TNFRSF13C (Proteintech, 22582-1, diluted 1:200), RASGRP4 (Abcam, 96293, diluted 1:100).

Techniques: Immunofluorescence, Staining, Expressing

Journal: Journal of cell science

Article Title: Tubular microdomains of Rab7-positive endosomes retrieve TrkA, a mechanism disrupted in Charcot-Marie-Tooth disease 2B.

doi: 10.1242/jcs.258559

Figure Lengend Snippet: Fig. 6. WASH1 is involved in late receptor tubulations. (A) Anti-GFP-conjugated beads were used to immunoprecipitate (IP) GFP, endophilinA1–GFP (A1–GFP), endophilinA2– GFP (A2–GFP) or endophilinA3–GFP (A3–GFP) with TrkA–RFP from co-transfected HEK293 cells (input on the left, IP on the right) in the presence or absence (NF) of 100 ng/ml NGF. (B) Anti-RFP-conjugated beads (or IgG control beads) were used to pull down EGFR–RFP with endophilinA1–GFP, endophilinA2–GFP or endophilinA3–GFP from co-transfected HEK293 cells in the presence or absence (NF) of 100 ng/ ml EGF (input on the left, IP on the right). Data in A and B are representative of three experiments. (C) Immunostaining of MEFs showing WASH1 on the rim of late Rab7 vacuoles when stimulated with NGF. Scale bar: 20 µm. (D) Immunostaining of MEFs showing WASH1 not localizing to late Rab7 vacuoles when stimulated with EGF. Scale bar: 20 µm. In C and D, boxes indicate regions shown as magnified images. Lines indicate transects shown in plot profiles. Nuclei are stained with DAPI. Images are representative of three experiments. (E) Colocalization of stained WASH1 and Rab7 increases in DRGs upon stimulation with NGF. Boxes indicate regions shown in magnified images on the right. Arrowheads indicate colocalization. Quantification shows mean±s.e.m. Pearson’s correlation coefficient. n=10–15 images per condition, the experiment was performed three times. **P=0.0046 (two-tailed, unpaired t-test). Scale bar: 10 µm. (F) EndophilinAs co- immunoprecipitate with WASH1 in lysates from HEK293 cells co-transfected with GFP-tagged endophilinAs and WASH1–mCherry. Anti-GFP- conjugated beads (or control IgG beads) were used for the IP [input and output (protein levels after incubation with conjugated beads) on the left, IP on the right]. Blots shown are representative of three experiments.

Article Snippet: Immunofluorescence was performed using the following antibodies: TrkA polyclonal rabbit antibody (Millipore, 06-574), EGFR (A10) mouse monoclonal antibody (Santa Cruz, sc-373746), p-TrkA Y794 polyclonal rabbit antibody (Millipore, ABN1383), pEGFR Y1068 monoclonal rabbit antibody (Cell Signaling Technologies, 3777), βIIItubulin mouse monoclonal antibody (Abcam, ab78078), WASH1 polyclonal rabbit antibody (Sigma, SAB4200372), endophilinA1 mouse monoclonal antibody (Santa Cruz, sc-374279), endophilinA2 mouse monoclonal antibody (Santa Cruz, sc-365704), endophilinA2 rabbit polyclonal antibody (Proteintech, 27014-1-AP), endophilinA3 mouse monoclonal antibody (Santa Cruz, sc-376592), Rab7 mouse monoclonal antibody (Cell Signaling Technologies, 95746), Rab7 polyclonal rabbit antibody (Synaptic Systems, 320 003).

Techniques: Transfection, Control, Immunostaining, Staining, Two Tailed Test, Incubation