pan cd44 mab  (R&D Systems)

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: (A) Schematic representation of CD44 protein structure. CD44 is extensively O -glycosylated, and alternative splicing generates multiple isoforms, with CD44v3 bearing a heparan sulfate chain that can mediate FGF binding. (B) Western blotting and (C) quantification of CD44 protein levels using a pan-CD44 antibody in TC28a2 wild-type, COSMC -/- , and C1GALT1 -/- cells, normalized to GAPDH (n = 3 independent experiments). (D) Reintroduction of hC1GALT1 restored cell surface CD44 levels in C1GALT1 -/- knockout cells. (E) Detection and quantification of cell surface CD44v3 levels in wild-type and mutant lines using an anti-CD44v3 antibody and flow cytometry. (F) Validation of TC28a2 CD44 -/- knockout cells via Western blot using a pan-CD44 antibody. Flow cytometry analyses of cell surface CD44v3 levels, (H) 3G10 binding, and (I) FGF1 and (J) FGF2 binding in wild-type and CD44 -/- cells. Data points are shown as mean ± SD (n ≥ 3 independent experiments); p-values were determined by one-way ANOVA with Tukey post-test (C-E), and student’s T-test (G-J) with **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: For the detection of other proteins and ligands, lifted cells were incubated in suspension for 30 min at 4 °C with 0.5 μg/mL mAb 10E4 (AMSBio #370255-1, Clone F58-10E4, 1:5000), 1 μg/mL mAb 3G10 (AMSBio, #370260-S, clone F69-3G10, 1:1000), 1 μg/mL mAb 2B6 (AMSBio # 270432-CS, Clone 2B6, 1:200), 0.1 μg/mL pan-CD44 mAb (Thermo #14-0441-82, Clone IM7, 1:1000), 0.5 μg/mL anti-CD44v3 mAb (R&D Systems #BBA11, Clone 3G5, 1:1000), 0.2 μg/mL anti-SDC1 mAb (Novus Biologicals #NB100-64980, Clone B-A38, 1:500), 0.5 μg/mL anti-SDC2 mAb (R&D Systems #MAB2965, Clone 305515, 1:1000), 80 nM biotin-FGF1, or 2.5 nM biotin-FGF2, respectively.

Techniques: Alternative Splicing, Binding Assay, Western Blot, Knock-Out, Mutagenesis, Flow Cytometry, Biomarker Discovery

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: (A) Flow cytometry analysis of cell surface CD44 levels in COSMC c6 and C1GALT1 t2 knockout clones. (B) Time course quantification of cell surface CD44 levels via flow cytometry prior to and after trypsinization (0-24 hours). Data is normalized to untreated wild-type cells. Data points are shown as mean ± SD (n = 3 independent experiments); p-values were determined by one-way ANOVA with Tukey post-test with **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. (C) CRISPR sgRNA targeting of human CD44 in TC28a2 chondrocytes. (C) Frameshift biallelic mutations in a clonal cell line for Exon 5 of CD44 were confirmed by Sanger sequencing.

Article Snippet: For the detection of other proteins and ligands, lifted cells were incubated in suspension for 30 min at 4 °C with 0.5 μg/mL mAb 10E4 (AMSBio #370255-1, Clone F58-10E4, 1:5000), 1 μg/mL mAb 3G10 (AMSBio, #370260-S, clone F69-3G10, 1:1000), 1 μg/mL mAb 2B6 (AMSBio # 270432-CS, Clone 2B6, 1:200), 0.1 μg/mL pan-CD44 mAb (Thermo #14-0441-82, Clone IM7, 1:1000), 0.5 μg/mL anti-CD44v3 mAb (R&D Systems #BBA11, Clone 3G5, 1:1000), 0.2 μg/mL anti-SDC1 mAb (Novus Biologicals #NB100-64980, Clone B-A38, 1:500), 0.5 μg/mL anti-SDC2 mAb (R&D Systems #MAB2965, Clone 305515, 1:1000), 80 nM biotin-FGF1, or 2.5 nM biotin-FGF2, respectively.

Techniques: Flow Cytometry, Knock-Out, Clone Assay, CRISPR, Sequencing

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: Flow cytometry analysis of (A) VVA lectin, (B) PNA lectin, (C) 3G10 antibody, (D) FGF1, (E) FGF2, and (F) pan-CD44 antibody binding in wild-type and knockout growth-plate-like chondroprogenitors (GPLCs). (G) Micromass cultures of GPLC wild-type, Cosmc -/- , and C1galt1 -/- cells at day 4 and day 7 after chondrogenic induction with insulin-transferrin-selenium (ITS). At each time point, cultures were fixed and stained with Alcian blue. (H) Quantification of Alcian blue staining confirmed visual data and confirmed statistical significance at Day 7. qPCR analysis of chondrogenic markers (I) Acan (J) Col2a1 , and (K) Sox9 in wild-type and knockout GPLC micromass cultures at Day 7. Data points are shown as mean ± SD (n ≥ 3 independent experiments); p-values were determined by one-way ANOVA with Tukey post-test with **** p < 0.0001, *** p < 0.001, ** p < 0.01.

Article Snippet: For the detection of other proteins and ligands, lifted cells were incubated in suspension for 30 min at 4 °C with 0.5 μg/mL mAb 10E4 (AMSBio #370255-1, Clone F58-10E4, 1:5000), 1 μg/mL mAb 3G10 (AMSBio, #370260-S, clone F69-3G10, 1:1000), 1 μg/mL mAb 2B6 (AMSBio # 270432-CS, Clone 2B6, 1:200), 0.1 μg/mL pan-CD44 mAb (Thermo #14-0441-82, Clone IM7, 1:1000), 0.5 μg/mL anti-CD44v3 mAb (R&D Systems #BBA11, Clone 3G5, 1:1000), 0.2 μg/mL anti-SDC1 mAb (Novus Biologicals #NB100-64980, Clone B-A38, 1:500), 0.5 μg/mL anti-SDC2 mAb (R&D Systems #MAB2965, Clone 305515, 1:1000), 80 nM biotin-FGF1, or 2.5 nM biotin-FGF2, respectively.

Techniques: Flow Cytometry, Binding Assay, Knock-Out, Staining

Journal: International journal of molecular sciences

Article Title: Apoptotic Caspases-3 and -7 Cleave Extracellular Domains of Membrane-Bound Proteins from MDA-MB-231 Breast Cancer Cells.

doi: 10.3390/ijms26083466

Figure Lengend Snippet: Figure 5. Immunological detection of caspase-mediated cleavages of extracellular proteins in normal (pH 7.4) and acidic (pH 6.0) environments. For the detection of extracellular cleavages, MDA-MB-231 cells were used. Red arrows indicate bands representing cleaved fragments, while black arrows indicate full-length proteins present in total cell lysates. (A) Detected caspase-3 or -7 extracellular cleavages of the selected targets (NRP-1, CD44, CSPG4) in an acidic (pH 6.0) environment in either DPBS or MES buffer. (B) Detection of caspase extracellular cleavages in a normal (pH 7.4) environment using either DPBS or HEPES buffer. NRP-1, neuropilin-1; CD44, CD44 antigen; CSPG4, chondroitin sulfate proteoglycan 4.

Article Snippet: Primary antibodies used for detection were polyclonal rabbit antibodies against cleaved caspase-3 (#9661, Cell Signaling, Danvers, MA, USA), polyclonal rabbit antibodies against cleaved caspase-7 (#9491, Cell Signaling, Danvers, MA, USA), polyclonal rabbit antibodies against β-actin (A2066, Sigma Aldrich, St. Louis, MO, USA), polyclonal sheep antibodies against human neuropilin-1 (#AF3870, R&D Systems, Minneapolis, MN, USA), monoclonal mouse antibodies against human CD44 (#3570, Cell Signaling, Danvers, MA, USA) and rabbit monoclonal antibodies against human CSPG4 (#43916, Cell Signaling, Danvers, MA, USA).

Techniques:

Journal: International journal of oral science

Article Title: Programmed death-ligand 1 regulates ameloblastoma growth and recurrence.

doi: 10.1038/s41368-025-00364-w

Figure Lengend Snippet: Fig. 4 The expression of proliferation-, stemness-, and p-EMT-related proteins is increased in human ameloblastoma. a Representative IHC images of proliferation markers (Ki-67 and PCNA) in OM (n = 16), OKC (n = 33), and AM (n = 60) tissues. Scale bar, 100 µm. b Quantification of H-scores for the proliferation markers Ki-67 and PCNA. c Representative IHC images of stemness markers (CD44, CD133, and ALDH1A1) in OM (n = 16), OKC (n = 33), and AM (n = 60) tissues. Scale bar, 100 µm. d Quantification of H-scores for the stemness markers CD44, CD133, and ALDH1A1. The data are presented as the means ± SDs. Statistical significance was assessed via two-tailed unpaired Student’s t test (b, d)

Article Snippet: The following primary antibodies were utilized in the study: anti-human PD-L1 (Cell Signaling Technology, #13684), anti-human CD44 (Cell Signaling Technology, #3570), anti-human/mouse CD133 (Proteintech, #18470-1-AP), anti-human/mouse ALDH1A1 (Proteintech, #60171- 1-Ig), anti-human LAMC2 (Abcam, #ab210959), anti-human LAMB3 (Abcam, #ab97765), anti-human PDPN (Cell Signaling Technology, #9047S), and anti-human/mouse GAPDH (ABclonal, #AC002).

Techniques: Expressing, Two Tailed Test

Journal: Cell communication and signaling : CCS

Article Title: Concomitant targeting of FLT3 and SPHK1 exerts synergistic cytotoxicity in FLT3-ITD + acute myeloid leukemia by inhibiting β-catenin activity via the PP2A-GSK3β axis.

doi: 10.1186/s12964-024-01774-9

Figure Lengend Snippet: Fig. 8 Combined SPHK1 inhibitor and TKI treatment suppresses β-catenin signaling, reducing β-catenin nuclear localization. (a) Western blot analysis of Molm13 cells after treatment with sorafenib (15 nM), SKI-II (20 µM), or both for 24 and 48 h. (b) Western blot analysis of primary FLT3-ITD+ AML blasts treated with sorafenib (2 µM), SKI-II (20 µM), or both for 48 h. (c) Western blot analysis of Molm13 and MV4-11 cells expressing shCtrl or shSPHK1 following sorafenib (15 or 30 nM) treatment for 24 h. (d) Confocal images and quantification of total CD44, total β-catenin, and nuclear β-catenin levels in Molm13 or MV4-11 cells treated with sorafenib (15 or 30 nM), SKI-II (20 µM), or both for 48 h. Scale bars indicate 10 μm. (e) β-catenin expression in total, cytoplas mic, and nuclear fractions was analyzed by western blot in Molm13 or MV4-11 cells treated with sorafenib (15 or 30 nM), SKI-II (20 µM), or both for 48 h, as determined via western blot. Data are presented as the mean ± SEM. Ctrl, control; Sor, sorafenib; Comb, combination. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: The cells were then stained with rabbit anti-human β-catenin and mouse anti-human CD44 primary antibodies (1:200 dilution; CST), followed by incubation with Alexa Fluor488-conjugated goat anti-rabbit and Alexa Fluor 594-conjugated goat anti-mouse secondary antibodies (1:5000 dilution; Abcam, Cambridge Biomedical Campus, Cambridge, UK).

Techniques: Western Blot, Expressing, Control