anti ku80  (Proteintech)

Journal: Nucleic Acids Research

Article Title: FOXN3 integrates the KU70/KU80/SREBP-1 complex to regulate lipid metabolism in non-alcoholic fatty liver disease

doi: 10.1093/nar/gkag171

Figure Lengend Snippet: FOXN3 and the KU70/KU80/SREBP-1 complex colocalize at the promoters of SREBP-1 response genes. ( A ) Mass spectrometry analysis of HEK293T cells transfected with Flag-tagged FOXN3 reveals the number of unique peptides associated with FOXN3. The representative peptide sequences of KU70 and KU80, which were immunoprecipitated by Flag-tagged FOXN3, are presented. ( B ) An anti-Flag Co-IP assay was performed in HEK293T cells transfected with the specified plasmids to investigate the association of Flag-tagged FOXN3 with the KU70/KU80/SREBP-1 complex (P: precursor of SREBP-1). ( C ) Anti-FOXN3 Co-IP analysis was performed in HepG2 cells to examine the endogenous association between FOXN3 and KU70/KU80/SREBP-1 complex. (P: precursor of SREBP-1; N: nuclear form of SREBP-1). ( D ) Anti-Flag Co-IP assays were performed in HepG2 cells to detect the association of Flag-tagged FOXN3 with the KU70/KU80/SREBP-1 complex following treatment with FFA (400 μM) at the indicated time points. The cells were treated with MG-132 (20 μM, 4 h) prior to collection (P: precursor of SREBP-1; N: nuclear form of SREBP-1). ( E ) Venn diagrams showing the overlapping peaks identified by anti-KU80, anti-FOXN3, and anti-SREBP-1 CUT&Tag analyses in HepG2 cells treated with FFA (400 μM, 24 h). ( F ) Venn diagrams displaying the number of genes co-targeted by KU80, FOXN3, and SREBP-1 based on CUT&Tag analyses in HepG2 cells treated with FFA (400 μM, 24 h). ( G ) Density distributions (normalized read densities) of the mapped reads from anti-KU80, anti-FOXN3, and anti-SREBP-1 CUT&Tag analyses at the transcription start site (TSS) are shown. ( H ) Genomic distribution of the transcriptional targets of KU80, FOXN3, and SREBP-1 in HepG2 cells treated with FFA (400 μM, 24 h), as determined via CUT&Tag data analysis. ( I ) The CUT&Tag data analysis in HepG2 cells treated with FFA (400 μM, 24 h) displays the binding profiles of KU80, FOXN3, and SREBP-1 to representative SREBP-1 response genes. The graphs represent the proportion of reads enriched in the peak region relative to every one million total reads. The blotting data D was quantified as the mean fold change from two independent experiments using ImageJ software and was analyzed using two-tailed Student’s t -tests; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies utilized were as follows: anti-SREBP-1 (14088-1-AP, Proteintech), anti-HA (51064-2-AP, Proteintech), anti-His (66005-1-Ig, Proteintech), anti-KU70 (10000-0-AP, Proteintech), anti-FOXN3 (25399-1-AP, Proteintech), anti-Flag (20543-1-AP, Proteintech), anti-KU70 (10723-1-AP, Proteintech), anti-KU80 (16389-1-AP, Proteintech), anti-β-actin (20536-1-AP, Cell Signaling Technology), anti-FASN (10624-2-AP, Proteintech), anti-SCD1 (28678-1-AP, Proteintech), anti-LDLR (10785-1-AP, Proteintech), anti-HMGCS1 (17643-1-AP, Proteintech), anti-SREBP-1 (66875-1-Ig, Proteintech, for IP), anti-rabbit IgG (30000-0-AP, Proteintech), and anti-FOXN3 (ab129453, Abcam).

Techniques: Mass Spectrometry, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Software, Two Tailed Test

Journal: Advanced Science

Article Title: APOL2 Stabilizes Ku80 to Confer NHEJ‐Mediated Radioresistance in Gastric Cancer

doi: 10.1002/advs.202506294

Figure Lengend Snippet: APOL2 binds to and stabilizes Ku80. A) Schematic diagram of plasmid construction to assess NHEJ activity. B‐C) Representative flow cytometry images (B) and quantitative analysis of relative GFP‐positive cell percentages (C) for I‐SceI‐infected and uninfected cells treated with the hEJ5‐GFP reporter system ( n = 3). D) Volcano plot showing significant differential expression patterns in NC versus APOL2‐OE (upregulated genes are in red; downregulated genes are in blue (|log2FC | ≥ 1 and q‐value ≤ 0.05). E) Representative immunofluorescence images demonstrating the colocalization of APOL2 (red) and Ku80 (green) upon IR. Nuclear DNA was counterstained with DAPI (blue). Scale bar, 10µm. F) Co‐IP experiments revealed the exogenous interaction between APOL2 and Ku80 in HEK293T cells. G) Diagram of plasmid constructs encoding full‐length (WT) and truncated APOL2 mutants. H) IP and Western blot analysis of HEK293T lysates from cells collected 48 h after transfection with the indicated expression vector and probed with the indicated antibodies. I) The protein expression levels of Ku80 and APOL2 in HEK293T cells transfected with Flag‐Ku80 and GFP‐APOL2 or empty vector plasmids were measured via Western blot assays. J) Protein expression levels of Ku80 in HEK293T cells after treatment with 50 µ m CQ or 10 µ m MG132. K‐L). The effect of CHX (100 µg mL −1 ) treatment in HGC27 cells stably transfected with APOL2 KO and vector plasmids (K), as well as in MKN1 cells stably transfected with GFP‐APOL2 and NC plasmids (L) ( n = 3). Statistical analysis was performed via two‐tailed unpaired Student's t‐tests (C) or two‐way ANOVA (L). Data was presented as mean ± SD; *** P < 0.001, **** P < 0.0001. All data are representative of three independent experiments.

Article Snippet: The membrane was incubated with APOL2 (Proteintech, #25925‐1‐AP), Ku80 (Proteintech, #16389‐1‐AP), ubiquitin antibodies (Proteintech, #10201‐2‐AP), Ku70 (Affinity, #AF0300), USP7 (Abcam, #ab108931), and β‐actin (Proteintech, #20536‐1‐AP) antibodies at 4°C overnight.

Techniques: Plasmid Preparation, Activity Assay, Flow Cytometry, Infection, Quantitative Proteomics, Immunofluorescence, Co-Immunoprecipitation Assay, Construct, Western Blot, Transfection, Expressing, Stable Transfection, Two Tailed Test

Journal: Advanced Science

Article Title: APOL2 Stabilizes Ku80 to Confer NHEJ‐Mediated Radioresistance in Gastric Cancer

doi: 10.1002/advs.202506294

Figure Lengend Snippet: APOL2 stabilizes Ku80 via USP7‐mediated deubiquitination. A) Ubiquitination assay of Ku80 in HEK293T cells co‐transfected with Flag‐Ku80, HA‐Ub, GFP‐APOL2, or empty vector plasmids following treatment with 10 µ m MG132 for 6 h. Total cell lysates were immunoprecipitated with anti‐Flag, followed by Western blot to detect ubiquitinated Ku80. B) Ubiquitination assay of Ku80 in HEK293T cells co‐transfected with HA‐Ub, HA‐Ub (complete Lys48 residue only), HA‐Ub (complete Lys63 residue only), GFP‐APOL2, or empty vector plasmids. Following 10 µ m MG132 treatment for 6 h, immunoprecipitation (IP) was performed using anti‐Flag antibody with subsequent Western blot analysis using ubiquitination antibody. C) Co‐IP assays in HEK293T cells co‐transfected with GFP‐APOL2 and Myc‐USP7 revealed exogenous interactions between APOL2 and Myc‐USP7. D) Co‐IP assays in HEK293T cells transfected with Myc‐USP7 and Flag‐Ku80 reveal exogenous interactions between Myc‐USP7 and Ku80. E) Ku80 protein expression levels in HEK293T cells transfected with Flag‐Ku80 and increasing concentrations of Myc‐USP7 (0–5 µg) or empty vector plasmids were detected by Western blot. F) The expression level of Ku80 mRNA in MKN1 or HGC27 cells co‐transfected with control or Myc‐USP7 overexpression plasmids was quantified by qRT‐PCR ( n = 3). G) Assessment of Ku80 protein stability using CHX chase assay. HGC27 cells stably expressing GFP‐APOL2 or empty vector (negative control) were treated with CHX (100 µg mL −1 ) for 0, 8, and 16 h. H) Ku80 ubiquitination was analyzed in HEK293T cells co‐transfected with Flag‐Ku80, HA‐Ub, and Myc‐USP7 or empty vector plasmids following treatment with 10 µ m MG132 for 6 h. I) Ubiquitination assay of Ku80 in HEK293T cells co‐transfected with GFP‐APOL2, si‐USP7, Flag‐Ku80, HA‐Ub, or empty vector plasmids and treated with 10 µ m MG132 for 6 h. Statistical analysis was performed via two‐tailed unpaired Student's t ‐tests. Data are presented as mean ± SD; ns: not significant. All data is representative of three independent experiments.

Article Snippet: The membrane was incubated with APOL2 (Proteintech, #25925‐1‐AP), Ku80 (Proteintech, #16389‐1‐AP), ubiquitin antibodies (Proteintech, #10201‐2‐AP), Ku70 (Affinity, #AF0300), USP7 (Abcam, #ab108931), and β‐actin (Proteintech, #20536‐1‐AP) antibodies at 4°C overnight.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Residue, Co-Immunoprecipitation Assay, Expressing, Control, Over Expression, Quantitative RT-PCR, Stable Transfection, Negative Control, Two Tailed Test

Journal: Advanced Science

Article Title: APOL2 Stabilizes Ku80 to Confer NHEJ‐Mediated Radioresistance in Gastric Cancer

doi: 10.1002/advs.202506294

Figure Lengend Snippet: APOL2 promotes NHEJ repair through Ku80. A) NHEJ reporter assay in APOL2‐OE cells. Cells were transfected with the hEJ5‐GFP reporter plasmid and indicated constructs (Vector+si‐NC, APOL2+si‐NC, and APOL2+si‐Ku80), followed by infection with I‐SceI adenovirus to induce DNA DSBs. NHEJ repair efficiency was quantified via flow cytometry analysis of GFP‐positive cells 48 h post‐infection ( n = 3). B) NHEJ reporter assay in APOL2‐KO cells. Experimental groups: shNC+vector, APOL2‐KO+vector, and APOL2‐KO+Ku80. Analysis was performed as in A). C) Comet assay in APOL2‐OE cells after 3 Gy IR. Representative images of comet assay and tail moment quantification of: i) Vector+si‐NC, ii) APOL2+si‐NC, and iii) APOL2+si‐Ku80 groups of MKN1 cells ( n = 15). Scale bar, 10 µm. D) Comet assay in APOL2‐KO cells after IR. Experimental groups: i) shNC+vector, ii) APOL2‐KO+vector, and iii) APOL2‐KO+Ku80 groups of HGC27 cells. Scale bar, 10 µm. Analysis was performed as described in C). E) γ‐H2AX foci formation in APOL2‐OE cells after 3 Gy irradiation. Representative immunofluorescence images showing γ‐H2AX (green) and DAPI (blue) in shNC+vector, APOL2‐KO+vector, and APOL2‐KO+Ku80 groups of cells (left). Quantitative analysis of γ‐H2AX foci per cell ( n = 40, right). Scale bar, 10 µm. F) γ‐H2AX foci formation in APOL2‐KO cells after 3 Gy irradiation. Experimental groups: i) Vector+si‐NC, ii) APOL2+si‐NC, iii) APOL2+si‐Ku80 groups. Scale bar, 10 µm. Analysis was performed as in E). All experiments were performed in triplicate. Error bars represent mean ± SD. Statistical analysis was performed via two‐tailed unpaired Student's t‐tests (A‐B, E‐F) or two‐way ANOVA (C‐D). Data was presented as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All data are representative of three independent experiments.

Article Snippet: The membrane was incubated with APOL2 (Proteintech, #25925‐1‐AP), Ku80 (Proteintech, #16389‐1‐AP), ubiquitin antibodies (Proteintech, #10201‐2‐AP), Ku70 (Affinity, #AF0300), USP7 (Abcam, #ab108931), and β‐actin (Proteintech, #20536‐1‐AP) antibodies at 4°C overnight.

Techniques: Reporter Assay, Transfection, Plasmid Preparation, Construct, Infection, Flow Cytometry, Single Cell Gel Electrophoresis, Irradiation, Immunofluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: APOL2 Stabilizes Ku80 to Confer NHEJ‐Mediated Radioresistance in Gastric Cancer

doi: 10.1002/advs.202506294

Figure Lengend Snippet: APOL2 confers radioresistance in vivo. A) Schematic of the PDX model construction and treatment protocol. B) Representative images of different groups of excised xenograft tumors 28 days after 8 Gy local RT at the endpoint. C‐D) Xenograft tumor growth curves (C) and endpoint weight (D) for each treatment group ( n = 5). E–H) Representative IHC images and quantitative analysis of Ki67, γ‐H2AX, Ku80 and p‐DNA‐PKcs in tumor tissue sections ( n = 5). E‐F: Scale bar, 50 µm. G‐H: Scale bar, 100 µm. Statistical analysis was performed via two‐tailed unpaired Student's t‐tests (D, F‐H) or two‐way ANOVA (C). Data was presented as mean ± SD; ** P < 0.01, *** P < 0.001, **** P < 0.0001. All data are representative of three independent experiments.

Article Snippet: The membrane was incubated with APOL2 (Proteintech, #25925‐1‐AP), Ku80 (Proteintech, #16389‐1‐AP), ubiquitin antibodies (Proteintech, #10201‐2‐AP), Ku70 (Affinity, #AF0300), USP7 (Abcam, #ab108931), and β‐actin (Proteintech, #20536‐1‐AP) antibodies at 4°C overnight.

Techniques: In Vivo, Two Tailed Test

Journal: Advanced Science

Article Title: APOL2 Stabilizes Ku80 to Confer NHEJ‐Mediated Radioresistance in Gastric Cancer

doi: 10.1002/advs.202506294

Figure Lengend Snippet: FN restores radiosensitivity by disrupting APOL2‐Ku80 interactions. A) Schematic diagram of the high‐throughput screening workflow of for 850 natural compounds in WT and APOL2‐KO cells. B) Scatter plot showing the 65 natural products initially screened and identified. Relative convergence was calculated as (normalized survival rate of KO group)/(normalized survival rate of the WT group) × 100%, where the survival rate values were first normalized to their respective untreated control groups (set to 100%). Each dot represents a drug, and the red dots respectively indicate the threshold data of sensitive drugs in APOL2‐KO cells. C) The relative cell viability of vector and APOL2‐KO cells treated with 65 candidate natural products for 48 h was quantified by CCK‐8 method. D) Bar graph showing the relative viability (%) of WT (vector) and APOL2‐KO cells treated with FN at the indicated concentrations (0, 25, 50, 75 µ m ) for 48 h, as determined by CCK‐8 method (n = 3). E) FN‐mediated disruption of patient‐derived GC organoids. Representative bright‐field images showing organoid structural integrity after 48 h treatment with different concentrations of FN (0‐50 µ m , left). Scale, 10 µm. Quantitative analysis of the organoid relative survival rate was measured by ImageJ. Data normalized to the control group (set as 100%). F) Co‐IP was used to detect the interaction between APOL2 and Ku80 in HEK293T cells treated with 50 µ m FN for 48 h. G) Statistical graphs of the colony formation in vector and APOL2‐KO cells after IR and FN treatment ( n = 3). H) Representative images of excised xenograft tumors at the endpoint (day 28 post‐treatment). Nude mice bearing subcutaneous tumors derived from WT or APOL2 KO GC cells were treated with: i) vehicle, ii) FN (20 mg/kg), (iii) RT (8 Gy), or (iv) combination therapy ( n = 5). I) Quantitative analysis of tumor weights. Excised tumors were weighed after treatments as described in H). J,K) Representative IHC staining images (J) and quantitative analysis (K) of Ki67, γ‐H2AX, and Ku80. Scale bar, 100 µm. Statistical analysis was performed via two‐tailed unpaired Student's t ‐tests (E) or two‐way ANOVA (D, I, K). Data was presented as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All data are representative of three independent experiments.

Article Snippet: The membrane was incubated with APOL2 (Proteintech, #25925‐1‐AP), Ku80 (Proteintech, #16389‐1‐AP), ubiquitin antibodies (Proteintech, #10201‐2‐AP), Ku70 (Affinity, #AF0300), USP7 (Abcam, #ab108931), and β‐actin (Proteintech, #20536‐1‐AP) antibodies at 4°C overnight.

Techniques: High Throughput Screening Assay, Control, Plasmid Preparation, CCK-8 Assay, Disruption, Derivative Assay, Co-Immunoprecipitation Assay, Immunohistochemistry, Two Tailed Test

Journal: Advanced Science

Article Title: APOL2 Stabilizes Ku80 to Confer NHEJ‐Mediated Radioresistance in Gastric Cancer

doi: 10.1002/advs.202506294

Figure Lengend Snippet: Schematic diagram of the mechanism by which APOL2 enhances radioresistance in GC. APOL2 stabilizes Ku80 through USP7‐mediated deubiquitination, thereby facilitating NHEJ‐mediated DNA repair and enhancing the radioresistance of GC. FN can suppress the interaction between APOL2 and Ku80, reducing radioresistance.

Article Snippet: The membrane was incubated with APOL2 (Proteintech, #25925‐1‐AP), Ku80 (Proteintech, #16389‐1‐AP), ubiquitin antibodies (Proteintech, #10201‐2‐AP), Ku70 (Affinity, #AF0300), USP7 (Abcam, #ab108931), and β‐actin (Proteintech, #20536‐1‐AP) antibodies at 4°C overnight.

Techniques: