ddr1 primary antibody  (Cell Signaling Technology Inc)

Journal: NPJ Precision Oncology

Article Title: Y chromosome-linked EIF1AY deletion drives sex differences in multiple myeloma

doi: 10.1038/s41698-026-01317-0

Figure Lengend Snippet: A Volcano plot of differentially expressed genes between normal macrophages (MΦs) and tumor-associated macrophages (TAMs) from GSE143583 . B Heatmap of the 381 differentially expressed genes between MΦs and TAMs; color intensity indicates expression. C WGCNA module-trait correlation heatmap; rows = module eigengenes, columns = macrophage subtypes. D Venn diagram showing overlap among differentially expressed genes, MEturquoise module genes, and membrane proteins from the Membranome database. E PPI network of genes in ( D ) generated via STRING; edges indicate predicted interactions. F DDR1 expression levels in MΦs and TAMs from GSE143583 . G DDR1 mRNA in THP-1-Mφ cultured with or without CM from MM cells, measured by qRT-PCR. H qRT-PCR analysis of IL-10, TGF-β, ARG1, and CD206 mRNA levels in THP-1-Mφ cultured with CM from MM cells overexpressing DDR1. I Flow cytometry analysis of CD206 expression in the indicated experimental groups. J qRT-PCR analysis of DDR1 mRNA expression in THP-1-Mφ cultured with or without CM from EIF1AY-overexpressing RPMI-8226 cells. K qRT-PCR analysis of DDR1 mRNA expression in THP-1-Mφ cultured with or without CM from EIF1AY-overexpressing U266 cells under the same experimental conditions. L Immunofluorescence (IF) analysis of DDR1 expression in THP-1-Mφ cultured with or without CM from EIF1AY-overexpressing MM cells. M Migration assay of THP-1-Mφ transduced to overexpress DDR1, cultured in CM from MM cells with or without EIF1AY overexpression (CM Vector or CM oe-EIF1AY). Recruited cells in the lower chamber were quantified to assess whether DDR1 overexpression in THP-1-Mφ rescues the migration impairment induced by EIF1AY expression in MM cells. N THP-1-Mφ pretreated with CM from EIF1AY-overexpressing or control MM cells were subsequently transduced with DDR1 and indirectly cocultured with MM cells in a transwell system. MM cell proliferation was then assessed to evaluate the effect of macrophage-derived signals. O qRT-PCR analysis of IL-4 and IL-13 mRNA levels in RPMI 8226 cells following EIF1AY overexpression. P ELISA analysis of IL-4 and IL-13 secretion in culture supernatants from EIF1AY-overexpressing MM cells. Q qRT-PCR analysis of DDR1 mRNA expression in THP-1-Mφ stimulated with or without IL-4 and IL-13.

Article Snippet: After fixation in 4% paraformaldehyde, cells were blocked with goat serum and subsequently incubated with DDR1 primary antibody (1:100; Cell Signaling Technology, #5583).

Techniques: Expressing, Membrane, Generated, Cell Culture, Quantitative RT-PCR, Flow Cytometry, Immunofluorescence, Migration, Over Expression, Plasmid Preparation, Control, Transduction, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: NPJ Precision Oncology

Article Title: Y chromosome-linked EIF1AY deletion drives sex differences in multiple myeloma

doi: 10.1038/s41698-026-01317-0

Figure Lengend Snippet: A Scatterplot showing positive correlation between RPS4Y1 and EIF1AY in the GSE6401 dataset. B Correlation validated in male MM patient samples. C qRT-PCR of EIF1AY mRNA in RPMI-8226 cells after RPS4Y1 knockdown. D qRT-PCR of EIF1AY mRNA in RPMI-8226 cells after RPS4Y1 overexpression. E Western blot of EIF1AY protein in RPMI-8226 cells after RPS4Y1 knockdown. F Western blot of EIF1AY protein in RPMI-8226 cells after RPS4Y1 overexpression. G qRT-PCR of IL-10, TGF-β, ARG1, and CD206 in THP-1-Mφ cultured with CM from RPS4Y1-overexpressing MM cells. H Western blot of CD206 in THP-1-Mφ treated as in ( G ). I qRT-PCR of DDR1 mRNA in THP-1-Mφ cultured with CM from RPS4Y1-overexpressing RPMI-8226 cells. J IF staining of DDR1 in RPS4Y1-overexpressing RPMI-8226 cells. K qRT-PCR of IL-4 mRNA in RPMI-8226 cells overexpressing RPS4Y1. L qRT-PCR of IL-13 mRNA in RPMI-8226 cells treated as in ( K ). M ELISA quantification of IL-4 in culture supernatants after RPS4Y1 overexpression. N ELISA quantification of IL-13 in culture supernatants after RPS4Y1 overexpression. O Structure-based modeling of the RPS4Y1–EIF1AY complex showing key interaction residues and predicted binding affinity. P Co-IP in RPMI-8226 cells with anti-EIF1AY to detect RPS4Y1. Q Co-IP in RPMI-8226 cells with anti-RPS4Y1 to detect EIF1AY. R Co-IP in U266 cells with anti-EIF1AY to detect RPS4Y1. S Co-IP in U266 cells with anti-RPS4Y1 to detect EIF1AY. T Co-IP in Flag-RPS4Y1 cells to detect EIF1AY. U Co-IP in Flag-EIF1AY cells to detect RPS4Y1. V THP-1-Mφ chemotactic migration assays were performed using CM derived from RPS4Y1-overexpressing MM cells, with or without EIF1AY knockdown. W The number of tumor cells was quantified following incubation with CM from polarized THP-1-Mφ induced by either RPS4Y1-overexpressing MM cells or RPS4Y1-overexpressing MM cells with EIF1AY knockdown.

Article Snippet: After fixation in 4% paraformaldehyde, cells were blocked with goat serum and subsequently incubated with DDR1 primary antibody (1:100; Cell Signaling Technology, #5583).

Techniques: Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Co-Immunoprecipitation Assay, Migration, Derivative Assay, Incubation

Journal: NPJ Precision Oncology

Article Title: Y chromosome-linked EIF1AY deletion drives sex differences in multiple myeloma

doi: 10.1038/s41698-026-01317-0

Figure Lengend Snippet: A Heatmap showing the differential expression of immune-related genes between male and female MM samples. Female samples are depicted in blue and male samples in red; darker shades indicate higher expression levels. B Volcano plot illustrating differentially expressed immune-related genes between male and female MM samples. Red dots represent genes upregulated in males, green dots denote downregulated genes, and black dots indicate genes with no significant expression difference. C Kaplan–Meier curve showing OS in MM patients with high versus low CD134 expression. D Kaplan–Meier curve showing event-free survival (EFS) in MM patients with high versus low CD134 expression. E Kaplan–Meier curve showing progression-free survival (PFS) in MM patients with high versus low CD134 expression. F CD134 mRNA expression in HDs and MM samples. G CD134 protein expression in HDs and MM samples. H CD206 expression in THP-1-Mφ cultured with CM from CD134-silenced MM cells was evaluated by Western blot. I Flow cytometry analysis of CD206 expression in THP-1-Mφ treated as indicated. J Quantification of CD206-positive THP-1-Mφ from ( I ). K IF analysis of DDR1 in THP-1-Mφ cultured with CM from CD134-deficient MM cells. L qRT-PCR analysis of DDR1 mRNA in THP-1-Mφ cultured with CM from CD134-deficient MM cells. M qRT-PCR analysis of IL-4 mRNA in RPMI-8226 cells following CD134 knockdown. N qRT-PCR analysis of IL-13 mRNA in RPMI-8226 cells following CD134 knockdown. O ELISA quantification of IL-4 secretion in MM cell supernatants following CD134 knockdown. P ELISA quantification of IL-13 secretion in MM cell supernatants following CD134 knockdown. Q Transwell migration assay of THP-1-Mφ overexpressing DDR1 cocultured with CD134-overexpressing MM cells to assess whether DDR1 rescues the inhibitory effect of CD134 on macrophage recruitment. R Quantification of migrated THP-1-Mφ from ( Q ). S THP-1-Mφ were pretreated with CM from CD134-overexpressing or control MM cells, followed by DDR1 overexpression. These macrophages were then indirectly cocultured with MM cells using a transwell system to evaluate the impact of macrophage-derived signals on MM cell proliferation.

Article Snippet: After fixation in 4% paraformaldehyde, cells were blocked with goat serum and subsequently incubated with DDR1 primary antibody (1:100; Cell Signaling Technology, #5583).

Techniques: Quantitative Proteomics, Expressing, Cell Culture, Western Blot, Flow Cytometry, Quantitative RT-PCR, Knockdown, Enzyme-linked Immunosorbent Assay, Transwell Migration Assay, Control, Over Expression, Derivative Assay

Journal: NPJ Precision Oncology

Article Title: Y chromosome-linked EIF1AY deletion drives sex differences in multiple myeloma

doi: 10.1038/s41698-026-01317-0

Figure Lengend Snippet: EIF1AY, a Y chromosome-encoded protein, forms a complex with RPS4Y1 to directly bind and stabilize CD134 mRNA, thereby sustaining CD134 expression in MM cells. CD134 signaling suppresses the secretion of IL-4 and IL-13, cytokines that normally induce DDR1 expression on macrophages and promote their polarization toward the tumor-supportive M2 phenotype. The RPS4Y1-EIF1AY-CD134 axis inhibits M2 macrophage polarization and recruitment, limiting MM cell proliferation. Loss of EIF1AY disrupts this axis, resulting in increased IL-4 and IL-13 secretion, upregulated DDR1 expression on macrophages, enhanced M2 polarization, and accelerated tumor progression. This feed-forward regulatory loop reveals a Y chromosome-linked immune mechanism underlying sex differences in MM and identifies EIF1AY as a potential target for precision immunotherapy in male patients.

Article Snippet: After fixation in 4% paraformaldehyde, cells were blocked with goat serum and subsequently incubated with DDR1 primary antibody (1:100; Cell Signaling Technology, #5583).

Techniques: Expressing

Journal: Journal of Medicinal Chemistry

Article Title: Cellular Context Influences Kinase Inhibitor Selectivity

doi: 10.1021/acs.jmedchem.5c02916

Figure Lengend Snippet: Structure of TPKI-41 and best available DDR1/2 probes.

Article Snippet: Total DDR1 blots were blocked in 5% dry milk in TBST for 2 h, washed 3 × 5 min with TBST and incubated overnight with mouse β-actin and rabbit DDR1 antibody (CST).

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Cellular Context Influences Kinase Inhibitor Selectivity

doi: 10.1021/acs.jmedchem.5c02916

Figure Lengend Snippet: Inhibition of DDR1 autophosphorylation by TPKI-39. (A) Western blotting analysis of TPKI-39 treatment of HeLa cells transiently transfected with cDNA encoding DDR1b isoform. Indicated samples were cotreated with collagen I and TPKI-39. DDR1-IN-1 and TPKI-41 were included as positive and negative controls, respectively. (B) ImageJ quantification of DDR1 Western blots. Data is reported as ratio of pDDR1 to total DDR1, normalized to activation by collagen in the absence of inhibitor. Samples are normalized to β-actin loading control. Results are representative of n = 3 independent experiments.

Article Snippet: Total DDR1 blots were blocked in 5% dry milk in TBST for 2 h, washed 3 × 5 min with TBST and incubated overnight with mouse β-actin and rabbit DDR1 antibody (CST).

Techniques: Inhibition, Western Blot, Transfection, Activation Assay, Control

Journal: Journal of Medicinal Chemistry

Article Title: Cellular Context Influences Kinase Inhibitor Selectivity

doi: 10.1021/acs.jmedchem.5c02916

Figure Lengend Snippet: Structure of TPKI-41 and best available DDR1/2 probes.

Article Snippet: Phospho-blots were blocked in 5% BSA in TBST for 2 h, washed 3 × 5 min with TBST and incubated overnight with mouse β-actin and rabbit DDR1 antibodies (CST).

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Cellular Context Influences Kinase Inhibitor Selectivity

doi: 10.1021/acs.jmedchem.5c02916

Figure Lengend Snippet: Inhibition of DDR1 autophosphorylation by TPKI-39. (A) Western blotting analysis of TPKI-39 treatment of HeLa cells transiently transfected with cDNA encoding DDR1b isoform. Indicated samples were cotreated with collagen I and TPKI-39. DDR1-IN-1 and TPKI-41 were included as positive and negative controls, respectively. (B) ImageJ quantification of DDR1 Western blots. Data is reported as ratio of pDDR1 to total DDR1, normalized to activation by collagen in the absence of inhibitor. Samples are normalized to β-actin loading control. Results are representative of n = 3 independent experiments.

Article Snippet: Phospho-blots were blocked in 5% BSA in TBST for 2 h, washed 3 × 5 min with TBST and incubated overnight with mouse β-actin and rabbit DDR1 antibodies (CST).

Techniques: Inhibition, Western Blot, Transfection, Activation Assay, Control

Journal: Cell Reports Medicine

Article Title: Drug screening in 3D microtumors reveals DDR1/2-MAPK12-GLI1 as a vulnerability in cancer-associated fibroblasts

doi: 10.1016/j.xcrm.2025.102357

Figure Lengend Snippet: Doramapimod unexpectedly targets DDR1/2 and MAPK12, regulating extracellular matrix gene expression in cancer-associated fibroblasts (A) Kinome profiling of doramapimod . Left: residual kinase activity for 370 kinases treated with 500 nM doramapimod using a radioactive ATP assay. Kinases with <20% residual activity are indicated in red. Right: bar chart highlighting top inhibited kinases. (B) CAF gene expression after kinase knockdown . Heatmap showing changes in ACTA2 and CXCL12 expression in breast cancer-derived CAFs following siRNA knockdown of doramapimod target kinases. (C) Plot showing qPCR analysis of CXCL12 expression in breast CAFs following siRNA-mediated knockdown of DDR1, DDR2, and MAPK12, with or without doramapimod treatment. Data represent mean ± SEM; n = 3 per group. Student’s t test, compared with DMSO control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. (D) Transcriptional changes in CAFs upon DDR1 , DDR2 , or MAPK12 depletion . Volcano plots showing DEGs (>2-fold, p < 0.05) after siRNA knockdown of DDR1 (left), DDR2 (middle), or MAPK12 (right) in primary breast CAFs. Downregulated genes are shown in blue and upregulated in red. n = 3 per group. (E) Pathway enrichment of downregulated genes following combined DDR1/2 and MAPK12 knockdown . Bar graph showing enrichment across Gene Ontology, Reactome, and KEGG pathways, with ECM-related processes highlighted in red. (F) Neutralization of CAFs’ growth stimulatory effect through depletion of DDR1/2 and MAPK12 kinase expression . Top: schematic of the experimental design showing breast CAFs treated with siRNAs against the indicated kinases, then co-cultured with 4T1 cancer cells labeled with nuclear GFP. Bottom: growth curve of 4T1 cells co-cultured with kinase depleted CAFs, displayed as Mean ± SEM. n = >3 in each group. (G) DDR1/2 enhances p38 phosphorylation . Western blot analysis showing elevated levels of phospho-p38 in CAFs overexpressing DDR1 or DDR2 compared to GFP control. Total p38 and β-actin are shown as loading controls. (H) Proposed model of doramapimod action . Doramapimod inhibits the DDR1/2–MAPK12 signaling axis, which drives ECM production in CAFs.

Article Snippet: Rabbit monoclonal DDR1 (D1G6) XP Antibody , Cell Signaling Technology , Cat#5583; RRID:AB_10694842.

Techniques: Gene Expression, Activity Assay, ATP Assay, Knockdown, Expressing, Derivative Assay, Control, Neutralization, Cell Culture, Labeling, Phospho-proteomics, Western Blot

Journal: Cell Reports Medicine

Article Title: Drug screening in 3D microtumors reveals DDR1/2-MAPK12-GLI1 as a vulnerability in cancer-associated fibroblasts

doi: 10.1016/j.xcrm.2025.102357

Figure Lengend Snippet: DDR1/2 and MAPK12 converge at GLI to regulate ECM production and support cancer cell growth (A) Gene overlap and expression analysis . Left: a Venn diagram showing the overlap of genes downregulated in response to knockdown of DDR1/2 and MAPK12 in CAFs. Right: a heatmap displaying changes in the expression of 7 genes commonly downregulated by depletion of DDR1/2 and MAPK12. The transcriptional factors, ECM proteins, and immune regulatory roles of these genes are highlighted. (B) Doramapimod reduces GLI1 nuclear localization in CAFs . Left: representative immunofluorescence images showing reduced nuclear GLI1 signal in CAFs following doramapimod treatment (1 μM for 48 h). Right: quantification of nuclear GLI1 intensity from at least 20 cells per group, indicating significantly reduced nuclear localization. Scale bars, 20 μm; ∗∗ p < 0.01; Student’s t test. (C) GLI1 transcriptional activity is inhibited by doramapimod , DDR1/2 , and MAPK12 knockdown . Left: luciferase reporter assay in pancreatic CAFs shows decreased GLI1 transcriptional activity upon treatment with doramapimod (1 μM) or GANT61 (1 μM). Right: similar reduction in GLI1 activity observed upon knockdown of DDR1, DDR2, or MAPK12. Data represent mean ± SEM from two-three biological replicates; ∗ p < 0.05 and ∗∗ p < 0.01; unpaired Student’s t test. (D) GLI1 inhibition downregulates ECM-associated pathways . Pathway enrichment plots based on RNA-seq of breast and pancreatic CAFs treated with GANT61. Genes involved in ECM regulation, including integrin signaling, collagen formation, and matrix remodeling, are significantly downregulated (adjusted p values indicated by color scale). (E) DDR1/2 promotes nuclear localization of GLI . Representative images showing that overexpression of DDR1/2 in normal human pancreatic fibroblasts promotes nuclear localization of phosphorylated p38 MAPK and GLI1. Scale bars, 20 μm. (F) Neutralization of CAFs' growth stimulatory effect by GLI depletion . Left: plot showing growth of GFP-labeled 4T1 cancer cells on CAFs with intact or depleted levels of GLI1, displayed as mean ± SEM. N = >3 in each group. Student’s t tests with Holm-Sidak correction. ∗∗ p < 0.01. Right: Gant61 treatment does not affect 4T1 cancer cell growth directly in serum-supported monoculture, but significantly reduces 4T1 cell growth when co-cultured with CAFs in serum-free condition, displayed as mean ± SEM. n indicates at least 3 replicates in each group. (G) M odel of the non-canonical hedgehog pathway in CAFs . Schematic illustrating DDR1/2-mediated activation of p38/MAPK12 and GLI drives ECM production in CAFs and supports cancer cell growth.

Article Snippet: Rabbit monoclonal DDR1 (D1G6) XP Antibody , Cell Signaling Technology , Cat#5583; RRID:AB_10694842.

Techniques: Expressing, Knockdown, Immunofluorescence, Activity Assay, Luciferase, Reporter Assay, Inhibition, RNA Sequencing, Over Expression, Neutralization, Labeling, Cell Culture, Activation Assay