human lc3b protein  (MedChemExpress)

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Co-Immunoprecipitation (Co-IP) with antibody to the FLAG epitope, followed by SDS-PAGE and immunoblotting with antibody to LC3 (endogenous, IgG: isotype control; Input: 5% lysate). Short/long: exposure time of the same membrane. b Co-IP with antibody to the LC3 epitope, followed by SDS-PAGE and immunoblotting showing endogenous DSS1. a , b IgG light chain-specific secondary antibody was used. c Glutathione-S-Transferase (GST)-pulldown analysis for DSS1-LC3B interaction using 50 ng recombinantly expressed human full-length LC3B and 1 mg GST/GST-DSS1 (GST: negative control; Purified LC3B: positive control; Glutathione agarose beads: 50 µL). d Cells were lysed with EDTA (10 mM) or not, followed by metal bath at 95 °C for 5 min and immunoblotting with antibody to DSS1. e GST-pulldown analysis showing the interaction of LC3 and DSS1 mutant using 50 ng recombinantly expressed human LC3 and 1 mg GST-DSS1 W27GW39GW43GF52A (Glutathione agarose beads: 50 µL). f Caki-1 cells were transfected with plasmids encoding HA-DSS1 W27GW39GW43GF52A . Cell lysates and cell immunoprecipitants were immunoprecipitated with antibody to the LC3 epitope and HA epitope, respectively. g Knockdown of DSS1 or treatment with MG-132 (20 μM, 6 h before harvest) elevates LC3 protein levels in HEK293T cells (control: siNC and/or 0.02% DMSO, error bar: mean ± SD, two-tailed Welch’s t-test). h Immunoblotting showing the indicated protein levels in HEK293T cells transfected with control or DSS1 siRNA, and/or treated with 0.02% DMSO or MG-132 (20 μM, 6 h before harvest). i Immunoblotting of cycloheximide (CHX, 50 μg/mL) chase assay in sh DSS1 cells compared to sh NC cells (error bar: mean ± SD, two-tailed Welch’s t-test). c –e , g–i The samples derived from the same experiment were run on parallel gels, with each gel probed for a different antibody. a – i n = 3 independent experiments. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: Purified human LC3B protein was purchased from MedChemExpress ® (HY- P70909 ).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, FLAG-tag, SDS Page, Western Blot, Control, Membrane, Negative Control, Purification, Positive Control, Mutagenesis, Transfection, Knockdown, Two Tailed Test, Derivative Assay

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Representative images of immunofluorescence showing the subcellular localization of DSS1 (magenta), LC3 (yellow), and PSMD3 (azure) in ACHN cells ( n = 3 independent experiments). Nuclei: DAPI (blue). Scale bar: 5 μm. b LC-MS/MS analysis of LC3 interactomes identifies E3 ligases potentially associated with LC3 ( n = 3 independent experiments; Proteome Discoverer 2.4, Thermo Scientific; False discovery rate <0.01). c The mRNA expression of TRIM25 in ccRCC patients from the TCGA-KIRC dataset ( n = 595 distinct samples, tumor vs. normal, two-tailed Welch’s test). Boxplot: Center line = median; box = 25th to 75th percentiles; whiskers = minima to maxima. d Representative images of immunohistochemistry analysis showing TRIM25 protein levels upregulated in ccRCC tissues vs. normal ( n = 66 distinct pairs; scale bar: 50 μm; error bar: mean (centre) ± SD; two-tailed paired t-test). e , f Immunoblotting showing TRIM25 knockdown increases LC3 levels, while TRIM25 overexpression reduces LC3 levels ( n = 3 independent experiments, error bar: mean ± SD, two-tailed Welch’s t-test). g Co-IP analysis in Caki-1 cells showing the interaction between endogenous TRIM25 and LC3 ( n = 3 independent experiments). h GST-pulldown analysis for TRIM25-LC3B interaction using 50 ng recombinantly expressed human full-length LC3B and 1 mg GST-TRIM25 ( n = 3 independent experiments; Glutathione agarose beads: 50 µL). Asterisk: GST-TRIM25. i Immunoblotting of HEK293T cells transfected with HA-Ub , pcDNA3.1, and Myc-LC3B (wildtype) or Myc-LC3B mutant plasmids ( n = 3 independent experiments). j Immunoblotting of HEK293T cells transfected with Myc-LC3B and HA-Ub -wildtype or HA-Ub -K48R/K63R mutant plasmids ( n = 3 independent experiments). k Recombinantly expressed human GST-TRIM25 was incubated with recombinant Ub, LC3B, UbcH5a/UbcH5c, and His-UBE1 in a reaction system containing MgATP at 37 °C for 4 h. Samples were analyzed by SDS-PAGE and immunoblotting with antibody to Ub epitope ( n = 3 independent experiments). As shown in the result of GST-TRIM25, the polyubiquitination of LC3B (LC3B-Ub) is shown only when all the components are present in the mix. e , f , h –j The samples derived from the same experiment were run on parallel gels, with each gel probed for a different antibody. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: Purified human LC3B protein was purchased from MedChemExpress ® (HY- P70909 ).

Techniques: Immunofluorescence, Liquid Chromatography with Mass Spectroscopy, Expressing, Two Tailed Test, Immunohistochemistry, Western Blot, Knockdown, Over Expression, Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Incubation, Recombinant, SDS Page, Derivative Assay

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Multiplex immunohistochemistry showing spatial distribution of DSS1-driven cells with a high epithelial-mesenchymal transition (EMT) feature (E-cad low , Vimentin high ) around vascular cells (CD31 + , arrows, n = 4 distinct patients). Scale bar: 40 μm. b SPP1 RNA and SPP1 protein levels in tissues of diverse cancer types (kidney cancer: RNA, n = 35 distinct cell lines, protein, n = 110 distinct samples) from the Human Protein Atlas (HPA, www.proteinatlas.org ). c SPP1 Protein levels in normal tissues from the HPA database (kidney, high expression in proximal tubules, n = 3 slides). d Pseudo-bulk RNA levels of SPP1 in epithelial and immune cell types. AvgExp, Average expression. e Protein abundance of SPP1 between tumor and normal tissues in Clinical Proteomic Tumor Analysis Consortium (CPTAC)-ccRCC dataset ( n = 194 distinct samples, two-tailed Mann-Whitney U test). Boxplot: Center line = median; box = 25th to 75th percentiles; whiskers = minima to maxima. f Spearman correlation between SPP1 and DSS1 mRNA expression (log 2 Count Per Million, two-tailed spearman’s rank correlation test). g Immunoblotting showing SPP1 protein levels in ccRCC cells (sh DSS1 vs. sh NC , n = 3 independent experiments, error bar: mean ± standard deviation [SD], two-tailed Welch’s t-test). The samples derived from the same experiment were run on parallel gels, with each gel probed for a different antibody. h Spearman correlation between CD68 expression and coexpression of SPP1 - ITGB1 in spots of sample sections from either tumor core or tumor-stromal interface (Li2022 spatial dataset, n = 13 sections from 10 distinct patients; error bar: mean ± SD). i Schematic diagram (by Figdraw.com) illustrates the proposed mechanistic model of DSS1-driven cells in ccRCC metastasis: DSS1 (oligomer) interacts with pro-LC3B or LC3B-I, promoting LC3B degradation via E3 ubiquitin ligase TRIM25-mediated Lys-63 (K63)-linked polyubiquitination at LC3B-K51, leading to impaired macroautophagic flux and p62 accumulation, TWIST1 stabilization and increased TWIST1 nuclear transport, promoting EMT activation. DSS1 highly expressed (DSS1 hi ) tumor cells were increased in late-stage tumors and linked to microvascular invasion within a vascularized invasive niche at the tumor-stromal interface, mediated by SPP1-ITGB1 interactions. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: Purified human LC3B protein was purchased from MedChemExpress ® (HY- P70909 ).

Techniques: Multiplex Assay, Immunohistochemistry, Expressing, Quantitative Proteomics, Two Tailed Test, MANN-WHITNEY, Western Blot, Standard Deviation, Derivative Assay, Ubiquitin Proteomics, Activation Assay

Journal: Cancers

Article Title: The Proapoptotic Action of Pyrrolidinedione–Thiazolidinone Hybrids towards Human Breast Carcinoma Cells Does Not Depend on Their Genotype

doi: 10.3390/cancers16162924

Figure Lengend Snippet: The concentration of LC3B in the MCF-7 ( A ) and MDA-MB-231 ( B ) cells after 24 h of incubation with Les-6287 and the reference drug (doxorubicin) at 1 µM, 1.5 µM, and 2 µM concentrations. Data are presented as M ± SD from three independent experiments performed in duplicate. * p < 0.05 compared to control (non-treated) cells.

Article Snippet: High-sensitivity human Beclin-1 SimpleStep ELISA Kit (Cat. No. ab254511), and high-sensitivity human SimpleStep ELISA Kits for MMP-2 (Cat. No. ab267813), MMP-9 (Cat. No. ab246539), and ICAM-1 (CD54, Cat. No. ab174445) were from Abcam (Cambridge, UK); LC3B ELISA Kit (Cat. No. E4774Hu) was from BT LAB (Shanghai, China).

Techniques: Concentration Assay, Incubation, Control