rabbit anti ddr1 antibody  (Huabio Inc)

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: The membrane was blocked with LICOR blocking buffer (PBS) for 1 h at ambient temperature, followed by primary antibody at 1:1000 in the blocking buffer at 4°C for 1 h. Primary antibodies used were as follows: p -DDR1/2 (R&D Systems, MAB25382), phospho-p38 MAPK (Cell Signaling Technology (CST), 4631), DDR1 (CST, 5583), DDR2 (CST, 25814), GLI1 (CST, 3538), p38 MAPK (CST, 9212), p38gamma MAPK (CST, 2307), and β-actin (Sigma, A1978).

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: The membrane was blocked with LICOR blocking buffer (PBS) for 1 h at ambient temperature, followed by primary antibody at 1:1000 in the blocking buffer at 4°C for 1 h. Primary antibodies used were as follows: p -DDR1/2 (R&D Systems, MAB25382), phospho-p38 MAPK (Cell Signaling Technology (CST), 4631), DDR1 (CST, 5583), DDR2 (CST, 25814), GLI1 (CST, 3538), p38 MAPK (CST, 9212), p38gamma MAPK (CST, 2307), and β-actin (Sigma, A1978).

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