atp7b antibody  (Novus Biologicals)

Journal: Chemical & Biomedical Imaging

Article Title: An Activity-Based Sensing Approach to Monitor Nanomaterial-Promoted Changes in Labile Metal Pools in Living Systems

doi: 10.1021/cbmi.5c00237

Figure Lengend Snippet: Copper-based nanoparticle internalization affects the regulation of cellular copper ion flux. (a) Western Blot analysis of ATP7B, CTR1, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) proteins in A549 cells treated with 6.25 μg Cu·mL –1 Cu–oxide, 3.125 μg Cu·mL –1 Cu–HCF, or vehicle control for 1 or 24 h. Quantified (b) CTR1 and (c) ATP7b expression after incubation with Cu–oxide or Cu–HCF for 1 or 24 h. Error bars denote SEM. Full gel images are posted in the Supporting Information .

Article Snippet: ATP7B antibody was purchased from Novus Biological (NB100–360) and used with a dilution of 1:1000.

Techniques: Western Blot, Control, Expressing, Incubation

Journal: Chemical & Biomedical Imaging

Article Title: An Activity-Based Sensing Approach to Monitor Nanomaterial-Promoted Changes in Labile Metal Pools in Living Systems

doi: 10.1021/cbmi.5c00237

Figure Lengend Snippet: Schematic depiction of a model showing the effects of copper-based nanoparticles on copper homeostasis and compensatory biochemical responses in cells. (a) A basal, physiological scenario where copper and intracellular redox status are under normal and regulated homeostatic control. (b) A high-copper perturbation scenario following nanoparticle internalization and copper ion leaching where a significant pool of labile Cu­(II) is generated. To overcome this acute or chronic aberrant exposure to excess of this metal and accompanying oxidative stress, cells sequester excess copper via GSH and NRF2 antioxidant pathways with concomitant upregulation of the copper ion export protein ATP7B to promote copper efflux and downregulation of the copper ion import protein CTR1 to block copper influx.

Article Snippet: ATP7B antibody was purchased from Novus Biological (NB100–360) and used with a dilution of 1:1000.

Techniques: Control, Generated, Blocking Assay

Journal: Biomedicines

Article Title: TRIM14 Regulation of Copper Homeostasis and Cuproptosis: A New Strategy to Overcome Chemoresistance in Glioblastoma

doi: 10.3390/biomedicines13123085

Figure Lengend Snippet: CuCl 2 Enhances TMZ Cytotoxicity on Glioblastoma Cells and Inhibits Proliferation and Invasion. ( A ) Flow cytometry analysis of apoptosis in U251 (upper row) and T98G (lower row) cells treated with different concentrations of CuCl 2 (0–6 μM). ( B ) Quantification of PI-positive cells in U251 and T98G cells after CuCl 2 (0–6 μM) treatment, showing that CuCl 2 alone has low toxicity ( n = 3). ( C ) Dose–response curves of U251 and T98G cell viability after treatment with different concentrations of TMZ (0–200 μM) ( n = 3). ( D ) Cell viability of U251 and T98G cells treated with TMZ (25 μM) alone or in combination with different concentrations of CuCl 2 (0–6 μM) ( n = 3). ( E ) Immunofluorescence staining of U251 cells after different treatments. Green represents DLAT, red represents the mitochondrial marker TOM20, and yellow indicates colocalization of both (63×). ( F , G ) Representative images and quantitative analysis of clonogenic assays for U251 and T98G cells under different treatments ( n = 3). ( H , I ) Representative images and quantification of Transwell invasion assays showing that the invasion ability significantly decreased in the combined treatment group ( n = 3) (20×). ( J ) CS1 fluorescence probe assay to detect Cu + levels in U251 and T98G cells, showing significant Cu + accumulation in the combined treatment group (100×). ( K , M ) Western blot and grayscale analysis showing the expression of copper transport and cuproptosis-related proteins (ATP7A, FDX1, Lipoylated-DLAT, SLC31A1, ATP7B) in T98G cells after treatment with TMZ (0, 25 μM) and CuCl 2 (0–6 μM) ( n = 3). ( L , N ) Western blot and grayscale analysis showing the expression of the same proteins in U251 cells treated with different concentrations of TMZ (0, 12.5, 25 μM) ( n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) were blocked with 5% non-fat milk at room temperature for 2 h. Primary antibodies were applied according to the manufacturer’s instructions and incubated at 4 °C for 12 h. Primary antibodies included ATP7A (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), ATP7B (ProteinTech Group, Chicago, IL, USA, 1:1000), TRIM14 (ProteinTech Group, Chicago, IL, USA, 1:500), β-actin (ProteinTech Group, Chicago, IL, USA, 1:20,000), FDX1 (ProteinTech Group, Chicago, IL, USA, 1:1000), PCNA (ProteinTech Group, Chicago, IL, USA, 1:5000), Vimentin (ProteinTech Group, Chicago, IL, USA, 1:20,000), E-cadherin (ProteinTech Group, Chicago, IL, USA, 1:20,000), p100 (ProteinTech Group, Chicago, IL, USA, 1:5000), p52 (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), SLC31A1 (ProteinTech Group, Chicago, IL, USA, 1:5000), Lip-DLAT (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), Lip-DLST (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), and others.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Marker, Fluorescence, Western Blot, Expressing

Journal: Biomedicines

Article Title: TRIM14 Regulation of Copper Homeostasis and Cuproptosis: A New Strategy to Overcome Chemoresistance in Glioblastoma

doi: 10.3390/biomedicines13123085

Figure Lengend Snippet: TMZ+CuCl 2 Combined Treatment Inhibits Tumor Growth in Mice and Alters Copper Homeostasis and EMT-Related Protein Expression. ( A ) Representative images of subcutaneous xenograft tumors in nude mice after different treatments. The tumor volume in the control group is larger, while the combined treatment group shows significantly smaller tumors. ( B ) Dynamic changes in tumor volume for each group, showing a significant inhibition of tumor growth in the combined treatment group ( n = 5). ( C ) Statistical analysis of tumor weight at the end of the experiment, with the combined treatment group showing significantly lower tumor weight compared to the control group ( n = 5). ( D ) Representative images of immunohistochemical staining of xenograft tumors showing the expression levels of proliferation-related protein PCNA and copper death-related proteins FDX1 and ATP7A. In the combined treatment group, the proportion of positive cells for PCNA, FDX1, and ATP7A is reduced (20×). ( E ) Western blot analysis of copper transport and copper deposition-related proteins (ATP7A, FDX1, Lipoylated-DLAT, SLC31A1, ATP7B) and proliferation/emt-related proteins (PCNA, vimentin, E-cadherin) in the control group, only TMZ group, only CuCl 2 group and combination treatment group. ( F ) Grayscale analysis of protein expression showing significant downregulation of ATP7A, FDX1, PCNA, and Vimentin, and upregulation of E-cadherin in the combined treatment group ( n = 3) (** p < 0.01, **** p < 0.0001, ns: not significant).

Article Snippet: PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) were blocked with 5% non-fat milk at room temperature for 2 h. Primary antibodies were applied according to the manufacturer’s instructions and incubated at 4 °C for 12 h. Primary antibodies included ATP7A (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), ATP7B (ProteinTech Group, Chicago, IL, USA, 1:1000), TRIM14 (ProteinTech Group, Chicago, IL, USA, 1:500), β-actin (ProteinTech Group, Chicago, IL, USA, 1:20,000), FDX1 (ProteinTech Group, Chicago, IL, USA, 1:1000), PCNA (ProteinTech Group, Chicago, IL, USA, 1:5000), Vimentin (ProteinTech Group, Chicago, IL, USA, 1:20,000), E-cadherin (ProteinTech Group, Chicago, IL, USA, 1:20,000), p100 (ProteinTech Group, Chicago, IL, USA, 1:5000), p52 (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), SLC31A1 (ProteinTech Group, Chicago, IL, USA, 1:5000), Lip-DLAT (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), Lip-DLST (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), and others.

Techniques: Expressing, Control, Inhibition, Immunohistochemical staining, Staining, Western Blot

Journal: Biomedicines

Article Title: TRIM14 Regulation of Copper Homeostasis and Cuproptosis: A New Strategy to Overcome Chemoresistance in Glioblastoma

doi: 10.3390/biomedicines13123085

Figure Lengend Snippet: TRIM14 Overexpression Reduces Cu + Accumulation and Weakens TMZ+CuCl 2 Antitumor Effects. ( A , B ) Western blot validation of TRIM14 overexpression efficiency ( n = 3). ( C , D ) Clonogenic assay: TRIM14 overexpression partially restores the inhibition of cell proliferation by TMZ+CuCl 2 ( n = 3). ( E , F ) Transwell invasion assay: TRIM14 overexpression significantly enhances cell invasion ability and weakens the inhibitory effect of TMZ+CuCl 2 ( n = 3) (10×). ( G ) CS1 fluorescence probe detection of Cu + levels, showing significant Cu + accumulation in the combined treatment group, while fluorescence signals are significantly reduced in the TRIM14 overexpression group (100×). ( H , I ) Western blot analysis of copper homeostasis and cuproptosis-related proteins, showing that TRIM14 overexpression reverses the downregulation of ATP7A, FDX1, and Lipoylated-DLAT, and the upregulation of SLC31A1, while restoring ATP7B expression ( n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) were blocked with 5% non-fat milk at room temperature for 2 h. Primary antibodies were applied according to the manufacturer’s instructions and incubated at 4 °C for 12 h. Primary antibodies included ATP7A (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), ATP7B (ProteinTech Group, Chicago, IL, USA, 1:1000), TRIM14 (ProteinTech Group, Chicago, IL, USA, 1:500), β-actin (ProteinTech Group, Chicago, IL, USA, 1:20,000), FDX1 (ProteinTech Group, Chicago, IL, USA, 1:1000), PCNA (ProteinTech Group, Chicago, IL, USA, 1:5000), Vimentin (ProteinTech Group, Chicago, IL, USA, 1:20,000), E-cadherin (ProteinTech Group, Chicago, IL, USA, 1:20,000), p100 (ProteinTech Group, Chicago, IL, USA, 1:5000), p52 (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), SLC31A1 (ProteinTech Group, Chicago, IL, USA, 1:5000), Lip-DLAT (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), Lip-DLST (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), and others.

Techniques: Over Expression, Western Blot, Biomarker Discovery, Clonogenic Assay, Inhibition, Transwell Invasion Assay, Fluorescence, Expressing

Journal: Biomedicines

Article Title: TRIM14 Regulation of Copper Homeostasis and Cuproptosis: A New Strategy to Overcome Chemoresistance in Glioblastoma

doi: 10.3390/biomedicines13123085

Figure Lengend Snippet: TRIM14 knockdown promotes TMZ+CuCl 2 -induced copper ion accumulation and cuproptosis. ( A , B ) Western blot validation of TRIM14 knockdown efficiency, showing a significant reduction in TRIM14 expression in the sh-TRIM14 group ( n = 3). ( C – F ) Western blot analysis of copper homeostasis- and cuproptosis-related proteins. TRIM14 knockdown resulted in downregulation of ATP7A, while ATP7B and SLC31A1 showed no significant changes. Under TMZ+CuCl 2 treatment, FDX1 expression was decreased, whereas lipoylated-DLAT and lipoylated-DLST were upregulated, indicating enhanced mitochondrial lipoylated protein aggregation and amplified cuproptosis ( n = 3). ( G ) CS1 fluorescence probe detection of intracellular Cu + levels, showing that TMZ+CuCl 2 treatment induced Cu + accumulation, which was further elevated in the TRIM14 knockdown group (100×) (** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) were blocked with 5% non-fat milk at room temperature for 2 h. Primary antibodies were applied according to the manufacturer’s instructions and incubated at 4 °C for 12 h. Primary antibodies included ATP7A (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), ATP7B (ProteinTech Group, Chicago, IL, USA, 1:1000), TRIM14 (ProteinTech Group, Chicago, IL, USA, 1:500), β-actin (ProteinTech Group, Chicago, IL, USA, 1:20,000), FDX1 (ProteinTech Group, Chicago, IL, USA, 1:1000), PCNA (ProteinTech Group, Chicago, IL, USA, 1:5000), Vimentin (ProteinTech Group, Chicago, IL, USA, 1:20,000), E-cadherin (ProteinTech Group, Chicago, IL, USA, 1:20,000), p100 (ProteinTech Group, Chicago, IL, USA, 1:5000), p52 (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), SLC31A1 (ProteinTech Group, Chicago, IL, USA, 1:5000), Lip-DLAT (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), Lip-DLST (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), and others.

Techniques: Knockdown, Western Blot, Biomarker Discovery, Expressing, Amplification, Fluorescence

Journal: Biomedicines

Article Title: TRIM14 Regulation of Copper Homeostasis and Cuproptosis: A New Strategy to Overcome Chemoresistance in Glioblastoma

doi: 10.3390/biomedicines13123085

Figure Lengend Snippet: Low Expression of ATP7B in Glioma Patients. ( A ) ATP7B expression in GBM samples. ( B ) ATP7B expression distribution based on patient race. ( C ) ATP7B expression distribution based on patient gender. ( D ) ATP7A expression in GBM based on patient age. ( E ) ATP7A expression in GBM based on patient TP53 mutation status. ( F – H ) Kaplan-Meier (KM) curves showing prognosis of patients with high or low ATP7B expression in the TCGA-GBM, CGGA, and TCGA-LGG databases (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) were blocked with 5% non-fat milk at room temperature for 2 h. Primary antibodies were applied according to the manufacturer’s instructions and incubated at 4 °C for 12 h. Primary antibodies included ATP7A (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), ATP7B (ProteinTech Group, Chicago, IL, USA, 1:1000), TRIM14 (ProteinTech Group, Chicago, IL, USA, 1:500), β-actin (ProteinTech Group, Chicago, IL, USA, 1:20,000), FDX1 (ProteinTech Group, Chicago, IL, USA, 1:1000), PCNA (ProteinTech Group, Chicago, IL, USA, 1:5000), Vimentin (ProteinTech Group, Chicago, IL, USA, 1:20,000), E-cadherin (ProteinTech Group, Chicago, IL, USA, 1:20,000), p100 (ProteinTech Group, Chicago, IL, USA, 1:5000), p52 (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), SLC31A1 (ProteinTech Group, Chicago, IL, USA, 1:5000), Lip-DLAT (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), Lip-DLST (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), and others.

Techniques: Expressing, Mutagenesis