anti chmp5 antibody  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: The ESCRT protein CHMP5 promotes T cell leukemia by enabling BRD4-p300-dependent transcription

doi: 10.1038/s41467-025-59504-9

Figure Lengend Snippet: a Hallmark pathway enrichment scores of differentially expressed genes (DEGs) (fold-change ≥ ±1.2; adjusted p < 0.05) between n = 3 control (CT) versus n = 3 CHMP5-depleted (KD) CUTLL1 cells after 5 days of selection with puromycin. p -value determined by Weighted Kolmogorov–Smirnov test and adjusted for multiple comparisons. FDR: false discovery rate. b GSEA plots of the Hallmark MYC-targets_V2 pathway (left) and MYC-targets_V1 pathway (right). p -value determined by Weighted Kolmogorov-Smirnov test and adjusted for multiple comparisons. NES: normalized enrichment score. n = 3 samples per group. c Western blot of the indicated proteins in CUTLL1 CT and KD whole cell lysates. Data are representative of 15 independent experiments. d MYC protein ( n = 10) and mRNA ( n = 12) expression relative to Actin and normalized to CT CUTLL1 cells. Data are mean ± SD of biological replicates pooled from 5 independent experiments. Student’s t-test, two-tailed. e Correlation between CHMP5 protein and MYC mRNA (left, n = 17) and MYC protein (right, n = 28) in CUTLL1 cells. Data points are biological replicates pooled from five independent experiments. Rho (R) and p -values determined by Pearson Correlation test. f Western blot of CT and KD CUTLL1 cells transduced with empty vector (Vector) or murine CHMP5 lentivirus (mCHMP5) for 48 h. Representative of 3 independent experiments. g Quantification of MYC protein and mRNA relative to Actin of cells from ( f ). qPCR data are 2 technical replicates (mRNA), and data are representative of 2 independent experiments. h Western blot of CUTLL1 cells transduced with 2 independent shRNAs targeting CHMP5 or MYC sequences and control (CT). Representative of 4 independent experiments. i mRNA expression of CHMP5 and MYC relative to ACTB and normalized to CT. Data are mean ± SD of 4 biological replicates. Statistics, one-way ANOVA, corrected for multiple comparisons using Tukey test. j CUTLL1 growth kinetics determined by trypan blue counting. Data are mean ± SD of cell numbers of 3 biological replicates. p -value determined by Two-way ANOVA. Representative of 2 independent experiments. k Extracellular acidification rate (ECAR, left), and oxygen consumption rate (OCR, right) kinetics. Data are presented as mean of 6 technical replicates from 2 biological replicates per group ( n = 12). 2-DG, 2-deoxyglucose; FCCP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone. l Basal ECAR and OCR values of indicated CUTLL1 cells from data in ( k ). Data points are 6 technical replicates from 2 biological replicates ( n = 12). Source data are provided as a Source Data file.

Article Snippet: Note that in some samples the anti-CHMP5 polyclonal antibody (ThermoFisher Scientific, #PA5-63303) can detect the two h an CHMP5 isoform: the major 219 amino acid (UniProt: Q9NZZ3-1) and a smaller 171 amino acid (UniProt: Q9NZZ3-2) isoform.

Techniques: Control, Selection, Western Blot, Expressing, Two Tailed Test, Transduction, Plasmid Preparation

Journal: Nature Communications

Article Title: The ESCRT protein CHMP5 promotes T cell leukemia by enabling BRD4-p300-dependent transcription

doi: 10.1038/s41467-025-59504-9

Figure Lengend Snippet: a Western blot of fractionated CUTLL1, MOLT-3, SUPT1, and patient derived xenograft (PDX) T-ALL cells. W, whole cell; C, cytoplasmic; and N, nuclear lysates. Nuclear CHMP5 band intensities relative to the cytoplasmic band are indicated. Representative of 4 independent experiments. b , c Immunofluorescence images of CUTLL1 ( b ) and T-ALL PDX ( c ) stained with anti-CHMP5 primary antibody followed by a secondary AF647 antibody (Red) and DAPI for nuclear staining (blue). Images are representative of 6 samples (3 CUTLL1, 3 PDX) from 3 independent experiments. Scale bars indicate 10 µm. d Schematic representation of the full length (FL) and NLS truncation mutant (ΔNLS) constructs of murine CHMP5. Created in BioRender. Umphred-Wilson, K. (2025) https://BioRender.com/dzfs72l . e Western blot of fractionated CUTLL1 cells transduced with FLAG-tagged full-length CHMP5 (FL) or ΔNLS mutant encoding lentivirus. Representative of 6 experiments. f Quantification of the nuclear/cytoplasmic ratio of FL-CHMP5 and ΔNLS-CHMP5 determined by densitometry. Data points are biological replicates from n = 6 independent experiments. Student’s t test, two-tailed. g Western blot of fractionated CUTLL1 cells transduced with CHMP5-HA subjected to immunoprecipitation with isotype (IgG) or anti-HA antibodies. Representative of 3 experiments. h Western blot of fractionated CUTLL1 cells subjected to immunoprecipitation with IgG or anti-BRD4 antibodies. Representative of 3 experiments. i Western blot of fractionated PDX T-ALL cells subjected to immunoprecipitation with IgG or anti-BRD4 antibodies. Representative of 2 experiments. j Anti-FLAG immunoprecipitation of BRD4-FLAG and CHMP5-His recombinant proteins. Representative of 2 experiments. k Nuclear fractionation of CUTLL1 cells into soluble and chromatin-bound fractions. Representative of 2 independent experiments. l Schematic of the MYC gene locus indicating ChIP-qPCR primer binding: A, enhancer; B, promoter; C, NDME (NOTCH-dependent MYC enhancer); D, BDME (BRD4-dependent MYC enhancer). m ChIP-qPCR of anti-BRD4 and CHMP5-HA at the MYC locus normalized to isotype (IgG). Data are 2 technical replicates, representative of 3 independent experiments. n ChIP-qPCR of anti-BRD4 and CHMP5-HA normalized to IgG from CUTLL1 cells treated with vehicle (DMSO) or 500 nM of JQ1 for 18 h. Data are 2 technical replicates, representative of 2 independent experiments. o Venn-diagram of BRD4 bound genes (determined by ChIP-seq GSE51800 ) and DEGs between CT and KD CUTLL1 cells (Fig. ). The BRD4 ChIP-seq dataset are available in the NCBI Gene Expression Omnibus database under accession code GSE51800 . p Pathway analysis of the overlapping BRD4-bound genes and DEGs between CT and KD CUTLL1 cells (CT, n = 3; KD, n = 3 samples each) in ( o ). p -value determined by Weighted Kolmogorov-Smirnov test and adjusted for multiple comparisons. Source data are provided as a Source Data file.

Article Snippet: Note that in some samples the anti-CHMP5 polyclonal antibody (ThermoFisher Scientific, #PA5-63303) can detect the two h an CHMP5 isoform: the major 219 amino acid (UniProt: Q9NZZ3-1) and a smaller 171 amino acid (UniProt: Q9NZZ3-2) isoform.

Techniques: Western Blot, Derivative Assay, Immunofluorescence, Staining, Mutagenesis, Construct, Transduction, Two Tailed Test, Immunoprecipitation, Recombinant, Fractionation, ChIP-qPCR, Binding Assay, ChIP-sequencing, Gene Expression

Journal: Nature Communications

Article Title: The ESCRT protein CHMP5 promotes T cell leukemia by enabling BRD4-p300-dependent transcription

doi: 10.1038/s41467-025-59504-9

Figure Lengend Snippet: a Pie chart of genome-wide Pol II binding in control (CT) and CHMP5-depleted (KD) CUTLL1 cells determined by ChIP-seq. b Metaplot of Pol II density at transcriptional start site (TSS) of all genes from ( a ). c Metaplot of Pol II density at the transcriptional end site (TES) of all genes from ( a ). Data in ( a – c ) are genome-wide Pol II signals and are representative of 2 biological replicates; shaded area along line graphs represent standard error. d Schematic for the calculation of Pol II traveling ratio. e , f Pol II traveling ratio for BRD4-bound ( n = 4138 genes) ( e ) and the Hallmark MYC-Target genes ( n = 118) from GSEA in Fig. ( f ). Kolmogorov–Smirnov test is two-sided and not adjusted for multiple comparisons. g Dot plot comparison of Pol II traveling ratios between CT and KD cells highlighting BRD4 and MYC-target genes. h Pol II binding tracks in CT and KD CUTLL1 cells at the MYC and XBP1 gene loci. 3’-end of genes are highlighted. i mRNA expression of MYC, XBP1 , and TCF7 in CUTLL1 CT and KD cells treated with DMSO (−) or JQ1 (+) for 18 h. Data are 2 technical replicates, representative of 3 experiments. j The ratio of pS5 at the TSS to pS2 at the TES at the MYC locus in CT and KD cells based on the ChIP qPCR in (Supplementary Fig. ). Data points are 2 technical replicates. k , l Metaplot and boxplot of H3K27ac density at TSS ( k ) and TES ( l ) of active genes determined by ChIP-seq. Data in ( k , l ) are calculated for H3K27ac signals at active genes and representative of 2 biological replicates; shaded area along line graphs represent standard error; boxplot limits are the 25 th and 75 th percentiles, with center line indicating the median. Whiskers extend to the minimum and maximum values. n = 101, Student’s t test, two-tailed. m Hockey stick plots of ranked genome-wide H3K27ac signals in CT (top) and KD (bottom) CUTLL1 cells. Positions of key T-ALL genes are highlighted. n BRD4 and H3K27ac ChIP-seq tracks at the MYC and TCF7 gene loci in CUTLL1 cells. BDME BRD4-dependent MYC enhancer, NDME NOTCH-dependent MYC enhancer, SE super-enhancer, TE typical enhancer. Source data are provided as a Source Data file.

Article Snippet: Note that in some samples the anti-CHMP5 polyclonal antibody (ThermoFisher Scientific, #PA5-63303) can detect the two h an CHMP5 isoform: the major 219 amino acid (UniProt: Q9NZZ3-1) and a smaller 171 amino acid (UniProt: Q9NZZ3-2) isoform.

Techniques: Genome Wide, Binding Assay, Control, ChIP-sequencing, Comparison, Expressing, ChIP-qPCR, Two Tailed Test

Journal: Nature Communications

Article Title: The ESCRT protein CHMP5 promotes T cell leukemia by enabling BRD4-p300-dependent transcription

doi: 10.1038/s41467-025-59504-9

Figure Lengend Snippet: a Immunoprecipitation with isotype (IgG) or anti-BRD4 antibody from CT and KD CUTLL1 nuclear lysate. Representative of 2 independent experiments. b Quantification of p300 bound to BRD4 from ( a ) calculated as p300-to-BRD4 ratio and normalized to control (CT) cells. Data are two biological replicates from independent experiments, normalized to control. c Recombinant BRD4, p300, and CHMP5 immunoprecipitation with anti-BRD4 antibody. Ratio of p300 bound to BRD4 (quantified by densitometry) relative to no-CHMP5 lane 6 is indicated. Data is representative of 2 independent experiments. d Recombinant p300-FLAG and CHMP5-His immunoprecipitation with anti-FLAG antibody. Representative of 2 experiments. e The predicted model of how CHMP5 mediates the p300-BRD4 interaction to promote histone acetylation and productive transcription, Created in BioRender. Umphred-Wilson, K. (2025) https://BioRender.com/w4989rg . f ChIP-qPCR of p300 at the MYC locus normalized to IgG in CT and KD CUTLL1 cells. Data are 2 biological replicates from independent experiments. g ChIP-qPCR of BRD4, p300, and H3K27ac at the MYC BDME in CUTLL1 cells treated with DMSO (−) or 500 nM of JQ1 (+) for 18 h. Data are 2 technical replicates, representative of 3 experiments. h RT-qPCR of MYC in CT and KD cells treated with 500 nM CCS1477 or 2 μM A485 for 18 h. Data are 2 technical replicates, representative of 2 experiments. i BRD4, p300, and H3K27ac ChIP-qPCR on the MYC BDME from the cells in ( h ). Data are 2 technical replicates, representative of 2 experiments. j Viability of parental CUTLL1 cells treated with CCS1477 or JQ1 plus 200 nM JQ1 (left) or 500 nM CCS1477 (right) for 72 h. Data are mean of 3 technical replicates, representative of 2 experiments. k IC 50 for CUTLL1 cell killing by CCS1477 and JQ1 combinations. IC 50 calculated by non-linear best-fit analysis. p-value calculated by 2-way ANOVA. FC, fold-change of DMSO vs +JQ1 or +CCS. l IC 50 of MOLT-3, CUTLL1, and T-ALL PDX treated with CCS1477, JQ1, or NEO2734. Source data are provided as a Source Data file.

Article Snippet: Note that in some samples the anti-CHMP5 polyclonal antibody (ThermoFisher Scientific, #PA5-63303) can detect the two h an CHMP5 isoform: the major 219 amino acid (UniProt: Q9NZZ3-1) and a smaller 171 amino acid (UniProt: Q9NZZ3-2) isoform.

Techniques: Immunoprecipitation, Control, Recombinant, ChIP-qPCR, Quantitative RT-PCR

Journal: Nature Communications

Article Title: The ESCRT protein CHMP5 promotes T cell leukemia by enabling BRD4-p300-dependent transcription

doi: 10.1038/s41467-025-59504-9

Figure Lengend Snippet: a CHMP5 mRNA expression in normal thymocytes ( n = 21) and primary T-ALL ( n = 57) samples (GSE33470, GSE33469). Student’s t test, two-tailed. The CHMP5 publicly available data used in this study are available in the NCBI Gene Expression Omnibus database under accession code GSE33470 and GSE33469 . b Western blot of indicated proteins in T cells from healthy donors ( n = 3) and primary human T-ALL samples ( n = 4). Representative of 3 experiments. c Quantification of CHMP5 protein relative to Actin from ( b ). Data presented as mean ± SD of biological replicates ( n = 3T cells, n = 4 T-ALL). Student’s t test, two-tailed. d Overall survival of pediatric T-ALL patients (TARGET T-ALL) expressing high (top 20%) and low (bottom 20%) levels of CHMP5. p = 0.017, Log-rank test. Low CHMP5 n = 50, High CHMP5 n = 47, All patients n = 240. The TARGET-TALL publicly available data used in this study are available in the NCBI database of Genotypes and Phenotypes under accession code phs000464.v7.p1 . e Viability of control (CT) and CHMP5-depleted (KD) CUTLL1, SUPT1, LOUCY cells treated with cytarabine (AraC) for 3 days. Data are presented as mean of 3 technical replicates and representative of 2 independent experiments. f IC 50 for AraC in CUTLL1, SUPT1, and LOUCY cells from ( e ). IC 50 calculated by non-linear best-fit analysis. p -value calculated by 2-way ANOVA. FC, fold-change in CT versus KD IC 50 . g Viability of CT and KD CUTLL1 cells treated with Compound E (Comp E) plus 100 nM dexamethasone (Dex) for 3 days. Data are presented as mean of 3 technical replicates. h IC 50 for Compound E in CT and KD CUTLL1 cells. IC 50 calculated by non-linear best-fit analysis. p-value calculated by 2-way ANOVA. FC, fold-change in CT versus KD IC 50 . i , j Western blot of fractionated CUTLL1 cells treated with vehicle (DMSO) or 1 μM dexamethasone (Dex) for 18 h and expression of NR3C1 in CUTLL1 cells from ( j ). qPCR data are 2 technical replicates and results are representative of 3 independent experiments. k KOPTK1 and MOLT-3 cells were transduced with vector or mCHMP5 lentivirus also encoding GFP. After 48 h, GFP + cells were sorted and cultured for 6-8 days with trypan blue counting. l , m Proliferation of control or mCHMP5-overexpressing MOLT-3 cells. Sorted GFP + cells (as in k ) were cultured with 100 nM JQ1 ( l ) or 1 nM Vincristine ( m ) for 6 days. Each data point in ( k – m ) are average cell numbers of 2 biological replicates. Cell growth curves were generated with linear regression analysis and p -statistics of differences between the linear regression slopes of the curves are indicated. Data are representative of 2 independent experiments. Source data are provided as a Source Data file.

Article Snippet: Note that in some samples the anti-CHMP5 polyclonal antibody (ThermoFisher Scientific, #PA5-63303) can detect the two h an CHMP5 isoform: the major 219 amino acid (UniProt: Q9NZZ3-1) and a smaller 171 amino acid (UniProt: Q9NZZ3-2) isoform.

Techniques: Expressing, Two Tailed Test, Gene Expression, Western Blot, Control, Transduction, Plasmid Preparation, Cell Culture, Generated

Journal: Nature Communications

Article Title: The ESCRT protein CHMP5 promotes T cell leukemia by enabling BRD4-p300-dependent transcription

doi: 10.1038/s41467-025-59504-9

Figure Lengend Snippet: a Kaplan–Meier survival curve of leukemia mice generated by injection of ICN1-NGFR-transduced Chmp5 f/f Cd4 -Cre - (WT) or Chmp5 f/f Cd4 -Cre + (KO) c-Kit-enriched bone marrow (BM) cells. p , Mantel–Cox log-rank test; n = 9 mice/group. b Spleen images and mean ± SD spleen weights. Student’s t test, two-tailed. WT: n = 9 mice, KO: n = 8 mice, from 2 independent experiments. c Wright-Giemsa staining of blood from leukemia mice at 4 weeks post-transplant. Scale bar = 20 µm. d Representative flow cytometry plot of CD45.2 and NGFR expression on BM (top) and spleen (bottom) cells at 4 weeks after BM transplantation. Mean (±SD) of CD45.2 + NGFR + cell frequency (%) and absolute cell numbers are shown in graphs (right). Sample size: frequency, WT, n = 12, KO n = 15; numbers, WT, n = 11, KO, n = 15 experimental mice per group. Student’s t test, two-tailed. e Hallmark pathway enrichment scores of DEGs (fold-change ≥1.2; adjusted p < 0.05) in splenic WT vs KO CD45.2 + NGFR + cells at 4 weeks post-transplant ( n = 3 mice per group). p -value determined by Weighted Kolmogorov–Smirnov test and adjusted for multiple comparisons. f GSEA plots of Hallmark MYC-target pathways from panel ( e ) ( n = 3 mice per group). p-value determined by Weighted Kolmogorov–Smirnov test and adjusted for multiple comparisons. g Heatmap of DEGs from the MYC-target pathways. All genes, have a p < 0.05. h MYC-GFP expression on CD45.2 + NGFR + cells from mice with percentage of MYC-GFP + cells indicated (left) and mean ± SD GFP mean fluorescence intensity (MFI) (right). 2-way ANOVA. WT: n = 4; KO: n = 6 mice. i Representative flow cytometry plot of CD34 versus MYC-GFP expression on CD45.2 + NGFR + cells with mean ± SD frequencies of each gate in BM (top) and spleen (bottom). 2-way ANOVA. WT, n = 4; KO, n = 6 mice. Source data are provided as a Source Data file.

Article Snippet: Note that in some samples the anti-CHMP5 polyclonal antibody (ThermoFisher Scientific, #PA5-63303) can detect the two h an CHMP5 isoform: the major 219 amino acid (UniProt: Q9NZZ3-1) and a smaller 171 amino acid (UniProt: Q9NZZ3-2) isoform.

Techniques: Generated, Injection, Two Tailed Test, Staining, Flow Cytometry, Expressing, Transplantation Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: Helicobacter pylori outer membrane vesicles mediate central tolerance in C57BL/6J mice offspring T cells via maternal-fetal transmission

doi: 10.3389/fimmu.2025.1522842

Figure Lengend Snippet: The primer sets used for the detection of murine genes by qRT-PCR analysis.

Article Snippet: Goat anti-CHMP5 antibody (GeneTex, GTX106692, 1:500 dilution), goat anti-NF-κB antibody (Abcam, ab32536, 1:1000 dilution), and goat anti-IKK-β antibody (Abcam, ab124957, 1:1000 dilution) were added to the membranes, with β-actin being used as control.

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Helicobacter pylori outer membrane vesicles mediate central tolerance in C57BL/6J mice offspring T cells via maternal-fetal transmission

doi: 10.3389/fimmu.2025.1522842

Figure Lengend Snippet: Induction by H.pylori OMVs of changes in signaling molecules related to T cell development in fetal mice thymus and their relationships. On day 0, the thymus of E15-16 fetal mice was stimulated with H.pylori OMVs: (A) , Western blot analysis showing the expression of CHMP5, IKK-β, and NF-κB; (B) , the expression levels of cytokines IL-7, IL-2, IL-4, and IFN-γ as determined by ELISA. (C) , After stimulation with H.pylori OMVs in the thymus of E15-16 fetal mice on day 3 of culture, qRT-PCR showing the expression of JNK, CHMP5 and Bcl-2 mRNA. Each independent experiment was repeated three times; results are presented as mean ± SD. Statistical tests were performed using an unpaired two-tailed t test. * P < 0.05, ** P < 0.01.

Article Snippet: Goat anti-CHMP5 antibody (GeneTex, GTX106692, 1:500 dilution), goat anti-NF-κB antibody (Abcam, ab32536, 1:1000 dilution), and goat anti-IKK-β antibody (Abcam, ab124957, 1:1000 dilution) were added to the membranes, with β-actin being used as control.

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Helicobacter pylori outer membrane vesicles mediate central tolerance in C57BL/6J mice offspring T cells via maternal-fetal transmission

doi: 10.3389/fimmu.2025.1522842

Figure Lengend Snippet: Possible mechanism of central tolerance of thymic T cells induced by H.pylori OMVs. (A) , During the positive selection phase, crosstalk might occur between the OMV activation pathway and the MHC pathway, which might regulate the stability of CHMP5 protein and promote the development of positively selected T cells. (B) , During the negative selection phase, crosstalk might occur between the OMV activation pathway and the TCR signaling pathway to promote T cell apoptosis. *P < 0.05, ** P < 0.01, *** P < 0.001, ns: no significance.

Article Snippet: Goat anti-CHMP5 antibody (GeneTex, GTX106692, 1:500 dilution), goat anti-NF-κB antibody (Abcam, ab32536, 1:1000 dilution), and goat anti-IKK-β antibody (Abcam, ab124957, 1:1000 dilution) were added to the membranes, with β-actin being used as control.

Techniques: Selection, Activation Assay

Journal: Molecular Medicine

Article Title: CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway

doi: 10.1186/s10020-024-00819-6

Figure Lengend Snippet: The expression of CHMP5 was decreased in OA human and mouse articular cartilages. ( A ) The protein content of CHMP5 was determined in normal and OA patient cartilages by western blot. ( B ) Safranin O-fast green staining images and OARSI score of articular cartilages from the sham and DMM mice at 8 weeks after surgery (Mann-Whitney test). Bar: 500 μm. ( C ) Real time PCR and ( D ) immunohistochemistry (IHC) showed the expression of CHMP5 in the mouse cartilages (Mann-Whitney test). Bar: 50 μm. ** P < 0.01. *** P < 0.001

Article Snippet: The membranes were blocked by 5% (M/V) skimmed milk and then incubated with the anti-rabbit primary antibody CHMP5 (1:1000), collagen-II (1:500), cleaved poly (ADP-ribose) polymerases (PARP; 1:500), cleaved caspase-3 (1:1000), aggrecan (1:400) from Affinity Biosciences (Changzhou, China), SOX9 (1:1000), IκBα (1:1000), p-p65 (1:1000), p65 (1:500), MMP13 (1:1000), matrix metallopeptidase 3 (MMP3; 1:2000), ADAMTS5 (1:500) from ABclonal (Wuhan, China), Histone H3 (1:5000) from Gene Tex (USA) as well as anti-mouse control GAPDH (1:10000) from proteintech (Wuhan, China) overnight at 4 °C.

Techniques: Expressing, Western Blot, Staining, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: Molecular Medicine

Article Title: CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway

doi: 10.1186/s10020-024-00819-6

Figure Lengend Snippet: The identification of human chondrocytes and verification of transfection efficiency. ( A ) The chondrocytes were identified by detecting the levels of collagen-II using immunofluorescence (IF) assay. Bar: 50 μm. ( B ) The infection efficiency was detected by real-time PCR and western blot 24 h after the adenovirus infection. Adenovirus-mediated CHMP5 overexpression (Ad-CHMP5) increased the levels of CHMP5, and ( C ) CHMP5 small hairpin RNA (Ad-shCHMP5) decreased the levels of CHMP5 in the chondrocytes. *** P < 0.001

Article Snippet: The membranes were blocked by 5% (M/V) skimmed milk and then incubated with the anti-rabbit primary antibody CHMP5 (1:1000), collagen-II (1:500), cleaved poly (ADP-ribose) polymerases (PARP; 1:500), cleaved caspase-3 (1:1000), aggrecan (1:400) from Affinity Biosciences (Changzhou, China), SOX9 (1:1000), IκBα (1:1000), p-p65 (1:1000), p65 (1:500), MMP13 (1:1000), matrix metallopeptidase 3 (MMP3; 1:2000), ADAMTS5 (1:500) from ABclonal (Wuhan, China), Histone H3 (1:5000) from Gene Tex (USA) as well as anti-mouse control GAPDH (1:10000) from proteintech (Wuhan, China) overnight at 4 °C.

Techniques: Transfection, Immunofluorescence, Infection, Real-time Polymerase Chain Reaction, Western Blot, Over Expression

Journal: Molecular Medicine

Article Title: CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway

doi: 10.1186/s10020-024-00819-6

Figure Lengend Snippet: CHMP5 suppressed IL-1β-stimulated pro-apoptosis in the chondrocytes. ( A ) Expression of CHMP5 in the chondrocytes treated with IL-1β for different times was tested by real time PCR and western blot. *** P < 0.001 versus the 0 h group. ### P < 0.001 versus the 6 h group. ( B ) CHMP5 overexpression restored but ( C ) CHMP5 knockdown exacerbated the repression of chondrocyte viability induced by IL-1β. ( D ) CHMP5 overexpression reversed IL-1β-activated pro-apoptotic effect. ( E ) CHMP5 silencing promoted the effect. ( F ) The expression of cleaved caspase-3 and cleaved PARP were detected by western blot. * P < 0.05. ** P < 0.01. *** P < 0.001

Article Snippet: The membranes were blocked by 5% (M/V) skimmed milk and then incubated with the anti-rabbit primary antibody CHMP5 (1:1000), collagen-II (1:500), cleaved poly (ADP-ribose) polymerases (PARP; 1:500), cleaved caspase-3 (1:1000), aggrecan (1:400) from Affinity Biosciences (Changzhou, China), SOX9 (1:1000), IκBα (1:1000), p-p65 (1:1000), p65 (1:500), MMP13 (1:1000), matrix metallopeptidase 3 (MMP3; 1:2000), ADAMTS5 (1:500) from ABclonal (Wuhan, China), Histone H3 (1:5000) from Gene Tex (USA) as well as anti-mouse control GAPDH (1:10000) from proteintech (Wuhan, China) overnight at 4 °C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Over Expression

Journal: Molecular Medicine

Article Title: CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway

doi: 10.1186/s10020-024-00819-6

Figure Lengend Snippet: CHMP5 constricted ECM degradation induced by IL-1β in the chondrocytes. ( A, B ) Western blot showed the levels of collagen II, aggrecan, SOX9, MMP13, MMP3 and ADAMTS5 in the chondrocytes. ( C, D ) IF staining of collagen II exhibited the protective effect of CHMP5 on ECM in the chondrocytes. Bar: 50 μm

Article Snippet: The membranes were blocked by 5% (M/V) skimmed milk and then incubated with the anti-rabbit primary antibody CHMP5 (1:1000), collagen-II (1:500), cleaved poly (ADP-ribose) polymerases (PARP; 1:500), cleaved caspase-3 (1:1000), aggrecan (1:400) from Affinity Biosciences (Changzhou, China), SOX9 (1:1000), IκBα (1:1000), p-p65 (1:1000), p65 (1:500), MMP13 (1:1000), matrix metallopeptidase 3 (MMP3; 1:2000), ADAMTS5 (1:500) from ABclonal (Wuhan, China), Histone H3 (1:5000) from Gene Tex (USA) as well as anti-mouse control GAPDH (1:10000) from proteintech (Wuhan, China) overnight at 4 °C.

Techniques: Western Blot, Staining

Journal: Molecular Medicine

Article Title: CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway

doi: 10.1186/s10020-024-00819-6

Figure Lengend Snippet: IL-1β-activated NF-κB pathway was restrained by CHMP5 in the chondrocytes. ( A ) The protein contents of IκBα, p-p65 and p65 were quantified in the chondrocytes by western blot. ( B ) CHMP5 reversed IL-1β-stimulated increase of p65 expression in the nucleus and decrease of p65 expression in the cytoplasm. ( C ) CHMP5 bound with IκBα and USP15 proteins in the chondrocytes. ( D ) CHMP5 decreased the ubiquitination of IκBα in the chondrocytes. ( E ) Silencing CHMP5 downregulated IκBα levels in the chondrocytes with the addition of IL-1β

Article Snippet: The membranes were blocked by 5% (M/V) skimmed milk and then incubated with the anti-rabbit primary antibody CHMP5 (1:1000), collagen-II (1:500), cleaved poly (ADP-ribose) polymerases (PARP; 1:500), cleaved caspase-3 (1:1000), aggrecan (1:400) from Affinity Biosciences (Changzhou, China), SOX9 (1:1000), IκBα (1:1000), p-p65 (1:1000), p65 (1:500), MMP13 (1:1000), matrix metallopeptidase 3 (MMP3; 1:2000), ADAMTS5 (1:500) from ABclonal (Wuhan, China), Histone H3 (1:5000) from Gene Tex (USA) as well as anti-mouse control GAPDH (1:10000) from proteintech (Wuhan, China) overnight at 4 °C.

Techniques: Western Blot, Expressing

Journal: Molecular Medicine

Article Title: CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway

doi: 10.1186/s10020-024-00819-6

Figure Lengend Snippet: CHMP5 mitigated OA progression in the DMM-mediated mice. ( A ) CHMP5 diminished mouse cartilage destruction (Mann-Whitney test). Bar: 500 μm. ( B-D ) CHMP5, Collagen II, and MMP-13 were examined by IHC assay (Mann-Whitney test). Bar: 50 μm. *P < 0.05. **P < 0.01

Article Snippet: The membranes were blocked by 5% (M/V) skimmed milk and then incubated with the anti-rabbit primary antibody CHMP5 (1:1000), collagen-II (1:500), cleaved poly (ADP-ribose) polymerases (PARP; 1:500), cleaved caspase-3 (1:1000), aggrecan (1:400) from Affinity Biosciences (Changzhou, China), SOX9 (1:1000), IκBα (1:1000), p-p65 (1:1000), p65 (1:500), MMP13 (1:1000), matrix metallopeptidase 3 (MMP3; 1:2000), ADAMTS5 (1:500) from ABclonal (Wuhan, China), Histone H3 (1:5000) from Gene Tex (USA) as well as anti-mouse control GAPDH (1:10000) from proteintech (Wuhan, China) overnight at 4 °C.

Techniques: MANN-WHITNEY

Journal: Molecular Medicine

Article Title: CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway

doi: 10.1186/s10020-024-00819-6

Figure Lengend Snippet: A schematic diagram showing that CHMP5 attenuates OA via inhibiting IL-1β-induced chondrocyte apoptosis and ECM degradation, involving NF-κB pathway

Article Snippet: The membranes were blocked by 5% (M/V) skimmed milk and then incubated with the anti-rabbit primary antibody CHMP5 (1:1000), collagen-II (1:500), cleaved poly (ADP-ribose) polymerases (PARP; 1:500), cleaved caspase-3 (1:1000), aggrecan (1:400) from Affinity Biosciences (Changzhou, China), SOX9 (1:1000), IκBα (1:1000), p-p65 (1:1000), p65 (1:500), MMP13 (1:1000), matrix metallopeptidase 3 (MMP3; 1:2000), ADAMTS5 (1:500) from ABclonal (Wuhan, China), Histone H3 (1:5000) from Gene Tex (USA) as well as anti-mouse control GAPDH (1:10000) from proteintech (Wuhan, China) overnight at 4 °C.

Techniques: