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mouse monoclonal antibody transferrin receptor  (Thermo Fisher)


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    Thermo Fisher mouse monoclonal antibody transferrin receptor
    ATP13A2 KD disrupts intracellular iron homeostasis in α-Syn-SH cells. We confirmed expression level of iron related genes for analysis of the effect of ATP13A2 on iron homeostasis. ( A – C ) mRNA expression of TfR ( A ), DMT1 ( B ), FPN ( C ) analyzed by qRT-PCR in α-Syn-SH cells with ATP13A2 KD. Data were normalized to the GAPDH level ( n = 4, biological replicates, TfR : NC = 1.0, siATP = 1.423, DMT1 : NC = 1.0, siATP = 1.362, FPN : NC = 1.0, siATP = 0.835). ( D ) Protein expression of TfR, DMT1, FPN, and IRP2 detected by Western blot in α-Syn-SH cells with ATP13A2 KD. (E–H) Quantification of ( D ). Data were normalized to β-actin levels ( n = 3, biological replicates, TfR: NC = 1.0, siATP = 1.648, DMT1: NC = 1.0, siATP = 0.971, FPN: NC = 1.0, siATP = 0.921, IRP2: NC = 1.0, siATP = 0.927). Each value represents the mean ± SEM. Student’s t-test was used to test the significance of differences (n.s. means not significant. * p < 0.05, ** p < 0.01, *** p < 0.001). siATP: siRNA targeting ATP13A2, NC: siRNA of negative control, TfR: <t>Transferrin</t> receptor, DMT1: Divalent Metal Transporter 1, FPN: Ferroportin, IRP2: Iron Regulatory protein 2.
    Mouse Monoclonal Antibody Transferrin Receptor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 14498 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 98 stars, based on 14498 article reviews
    mouse monoclonal antibody transferrin receptor - by Bioz Stars, 2026-02
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    1) Product Images from "Disruption of intracellular iron homeostasis through mitochondrial dysfunction associated with suppression of ATP 13A2 expression"

    Article Title: Disruption of intracellular iron homeostasis through mitochondrial dysfunction associated with suppression of ATP 13A2 expression

    Journal: Scientific Reports

    doi: 10.1038/s41598-026-35368-x

    ATP13A2 KD disrupts intracellular iron homeostasis in α-Syn-SH cells. We confirmed expression level of iron related genes for analysis of the effect of ATP13A2 on iron homeostasis. ( A – C ) mRNA expression of TfR ( A ), DMT1 ( B ), FPN ( C ) analyzed by qRT-PCR in α-Syn-SH cells with ATP13A2 KD. Data were normalized to the GAPDH level ( n = 4, biological replicates, TfR : NC = 1.0, siATP = 1.423, DMT1 : NC = 1.0, siATP = 1.362, FPN : NC = 1.0, siATP = 0.835). ( D ) Protein expression of TfR, DMT1, FPN, and IRP2 detected by Western blot in α-Syn-SH cells with ATP13A2 KD. (E–H) Quantification of ( D ). Data were normalized to β-actin levels ( n = 3, biological replicates, TfR: NC = 1.0, siATP = 1.648, DMT1: NC = 1.0, siATP = 0.971, FPN: NC = 1.0, siATP = 0.921, IRP2: NC = 1.0, siATP = 0.927). Each value represents the mean ± SEM. Student’s t-test was used to test the significance of differences (n.s. means not significant. * p < 0.05, ** p < 0.01, *** p < 0.001). siATP: siRNA targeting ATP13A2, NC: siRNA of negative control, TfR: Transferrin receptor, DMT1: Divalent Metal Transporter 1, FPN: Ferroportin, IRP2: Iron Regulatory protein 2.
    Figure Legend Snippet: ATP13A2 KD disrupts intracellular iron homeostasis in α-Syn-SH cells. We confirmed expression level of iron related genes for analysis of the effect of ATP13A2 on iron homeostasis. ( A – C ) mRNA expression of TfR ( A ), DMT1 ( B ), FPN ( C ) analyzed by qRT-PCR in α-Syn-SH cells with ATP13A2 KD. Data were normalized to the GAPDH level ( n = 4, biological replicates, TfR : NC = 1.0, siATP = 1.423, DMT1 : NC = 1.0, siATP = 1.362, FPN : NC = 1.0, siATP = 0.835). ( D ) Protein expression of TfR, DMT1, FPN, and IRP2 detected by Western blot in α-Syn-SH cells with ATP13A2 KD. (E–H) Quantification of ( D ). Data were normalized to β-actin levels ( n = 3, biological replicates, TfR: NC = 1.0, siATP = 1.648, DMT1: NC = 1.0, siATP = 0.971, FPN: NC = 1.0, siATP = 0.921, IRP2: NC = 1.0, siATP = 0.927). Each value represents the mean ± SEM. Student’s t-test was used to test the significance of differences (n.s. means not significant. * p < 0.05, ** p < 0.01, *** p < 0.001). siATP: siRNA targeting ATP13A2, NC: siRNA of negative control, TfR: Transferrin receptor, DMT1: Divalent Metal Transporter 1, FPN: Ferroportin, IRP2: Iron Regulatory protein 2.

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Negative Control

    Protective effect of ATP13A2 KD in α-Syn-SH cells by inhibiting iron influx. Apo Transferrin or Glibenclamide (GBD) were administered for 24 h after ATP13A2 KD in α-Syn-SH cells. ( A ) Atomic absorption spectrometry for evaluation of total intracellular iron level in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD. Quantification of iron content per 1.0 × 10 5 cells ( n = 3, biological replicates, NC = 1.579, NC apo-Tf = 1.645, siATP = 2.671, siATP apo-Tf = 1.984). ( B ) Staining of RhoNox-4 for evaluation of intracellular Fe 2+ level in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD (Red: Rho-Nox4, Blue: Hoechst) (Scale bar, 10 μm). ( C ) Quantification of (B) ( n = 5, biological replicates, Cell number of NC for each biological replicate: 156, 89, 93, 94, 116, Cell number of NC apo-Tf for each biological replicate: 146, 101, 117, 91, 76, Cell number of siATP for each biological replicate: 90, 134, 94, 108, 126, Cell number of siATP apo-Tf for each biological replicate: 135, 103, 68, 120, 112, NC = 1.0, NC apo-Tf = 1.069, siATP = 1.510, siATP apo-Tf = 1.121). ( D ) MitoSOX dye for detection of mitochondrial ROS in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD (Red: MitoSOX, Blue: Hoechst) (Scale bar, 20 μm). ( E ) Quantification of (D) ( n = 4, biological replicates, Cell number of NC for each biological replicate: 203, 180, 99, 163, Cell number of NC apo-Tf for each biological replicate: 236, 223, 208, 199, Cell number of siATP for each biological replicate: 187, 188, 175, 145, Cell number of siATP for each biological replicate: 187, 188, 175, 145, Cell number of siATP apo-Tf for each biological replicate: 162, 181, 173, 166, NC = 1.0, NC apo-Tf = 1.013, siATP = 2.729, siATP apo-Tf = 1.552). ( F ) Cell viability was measured by CCK-8 assay in α-Syn-SH cells treated with apo Transferrin for 24 h (0.01, 0.10, 0.50, or 1.00 mg/mL) after ATP13A2 KD ( n = 4, biological replicates, NC = 100, siATP = 78.34, siATP apo-Tf 0.01 mg/mL = 91.84, siATP apo-Tf 0.10 mg/mL = 102.3, siATP apo-Tf 0.50 mg/mL = 105.0, siATP apo-Tf 1.0 mg/mL = 101.8). ( G ) Atomic absorption spectrometry in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD. Quantification of iron content per 1.0 × 10 5 cells ( n = 3, biological replicates, NC = 1.276, NC GBD = 1.130, siATP = 1.882, siATP GBD = 1.268). ( H ) Staining of RhoNox-4 in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD (Red: Rho-Nox4, Blue: Hoechst) (Scale bar, 10 μm). ( I ) Quantification of (H) ( n = 5, biological replicates, Cell number of NC for each biological replicate: 73, 52, 60, 77, 54, Cell number of NC GBD for each biological replicate: 62, 86, 99, 96, 94, Cell number of siATP for each biological replicate: 123, 65, 80, 115, 97, Cell number of siATP GBD for each biological replicate: 82, 64, 81, 127, 95, NC = 1.0, NC GBD = 1.073, siATP = 1.849, siATP GBD = 1.126). ( J ) MitoSOX dye in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD (Red: MitoSOX, Blue: Hoechst) (Scale bar, 20 μm). ( K ) Quantification of (J) ( n = 4, biological replicates, Cell number of NC for each biological replicate: 121, 119, 153,128, Cell number of NC GBD for each biological replicate: 115, 158, 125, 139, Cell number of siATP for each biological replicate: 106, 109, 83, 99, Cell number of siATP GBD for each biological replicate: 119, 122, 128, 97, NC = 1.0, NC GBD = 0.904, siATP = 2.325, siATP GBD = 1.839). ( L ) Cell viability was measured by CCK-8 assay in α-Syn-SH cells treated with Glibenclamide for 24 h (3.0, 10.0, or 30.0 µM) after ATP13A2 KD ( n = 6, biological replicates, NC = 100, NC GBD 10 µM = 118.1, siATP = 73.66, siATP GBD 3 µM = 87.66, siATP GBD 10 µM = 94.94, siATP GBD 30 µM = 95.94). Each value represents the mean ± SEM. An ANOVA, followed by the Bonferroni/Dunn post-hoc test was used to test the significance of differences (n.s. not significant. * p < 0.05, ** p < 0.01, *** p < 0.001). siATP: siRNA targeting ATP13A2, NC: siRNA of negative control.
    Figure Legend Snippet: Protective effect of ATP13A2 KD in α-Syn-SH cells by inhibiting iron influx. Apo Transferrin or Glibenclamide (GBD) were administered for 24 h after ATP13A2 KD in α-Syn-SH cells. ( A ) Atomic absorption spectrometry for evaluation of total intracellular iron level in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD. Quantification of iron content per 1.0 × 10 5 cells ( n = 3, biological replicates, NC = 1.579, NC apo-Tf = 1.645, siATP = 2.671, siATP apo-Tf = 1.984). ( B ) Staining of RhoNox-4 for evaluation of intracellular Fe 2+ level in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD (Red: Rho-Nox4, Blue: Hoechst) (Scale bar, 10 μm). ( C ) Quantification of (B) ( n = 5, biological replicates, Cell number of NC for each biological replicate: 156, 89, 93, 94, 116, Cell number of NC apo-Tf for each biological replicate: 146, 101, 117, 91, 76, Cell number of siATP for each biological replicate: 90, 134, 94, 108, 126, Cell number of siATP apo-Tf for each biological replicate: 135, 103, 68, 120, 112, NC = 1.0, NC apo-Tf = 1.069, siATP = 1.510, siATP apo-Tf = 1.121). ( D ) MitoSOX dye for detection of mitochondrial ROS in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD (Red: MitoSOX, Blue: Hoechst) (Scale bar, 20 μm). ( E ) Quantification of (D) ( n = 4, biological replicates, Cell number of NC for each biological replicate: 203, 180, 99, 163, Cell number of NC apo-Tf for each biological replicate: 236, 223, 208, 199, Cell number of siATP for each biological replicate: 187, 188, 175, 145, Cell number of siATP for each biological replicate: 187, 188, 175, 145, Cell number of siATP apo-Tf for each biological replicate: 162, 181, 173, 166, NC = 1.0, NC apo-Tf = 1.013, siATP = 2.729, siATP apo-Tf = 1.552). ( F ) Cell viability was measured by CCK-8 assay in α-Syn-SH cells treated with apo Transferrin for 24 h (0.01, 0.10, 0.50, or 1.00 mg/mL) after ATP13A2 KD ( n = 4, biological replicates, NC = 100, siATP = 78.34, siATP apo-Tf 0.01 mg/mL = 91.84, siATP apo-Tf 0.10 mg/mL = 102.3, siATP apo-Tf 0.50 mg/mL = 105.0, siATP apo-Tf 1.0 mg/mL = 101.8). ( G ) Atomic absorption spectrometry in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD. Quantification of iron content per 1.0 × 10 5 cells ( n = 3, biological replicates, NC = 1.276, NC GBD = 1.130, siATP = 1.882, siATP GBD = 1.268). ( H ) Staining of RhoNox-4 in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD (Red: Rho-Nox4, Blue: Hoechst) (Scale bar, 10 μm). ( I ) Quantification of (H) ( n = 5, biological replicates, Cell number of NC for each biological replicate: 73, 52, 60, 77, 54, Cell number of NC GBD for each biological replicate: 62, 86, 99, 96, 94, Cell number of siATP for each biological replicate: 123, 65, 80, 115, 97, Cell number of siATP GBD for each biological replicate: 82, 64, 81, 127, 95, NC = 1.0, NC GBD = 1.073, siATP = 1.849, siATP GBD = 1.126). ( J ) MitoSOX dye in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD (Red: MitoSOX, Blue: Hoechst) (Scale bar, 20 μm). ( K ) Quantification of (J) ( n = 4, biological replicates, Cell number of NC for each biological replicate: 121, 119, 153,128, Cell number of NC GBD for each biological replicate: 115, 158, 125, 139, Cell number of siATP for each biological replicate: 106, 109, 83, 99, Cell number of siATP GBD for each biological replicate: 119, 122, 128, 97, NC = 1.0, NC GBD = 0.904, siATP = 2.325, siATP GBD = 1.839). ( L ) Cell viability was measured by CCK-8 assay in α-Syn-SH cells treated with Glibenclamide for 24 h (3.0, 10.0, or 30.0 µM) after ATP13A2 KD ( n = 6, biological replicates, NC = 100, NC GBD 10 µM = 118.1, siATP = 73.66, siATP GBD 3 µM = 87.66, siATP GBD 10 µM = 94.94, siATP GBD 30 µM = 95.94). Each value represents the mean ± SEM. An ANOVA, followed by the Bonferroni/Dunn post-hoc test was used to test the significance of differences (n.s. not significant. * p < 0.05, ** p < 0.01, *** p < 0.001). siATP: siRNA targeting ATP13A2, NC: siRNA of negative control.

    Techniques Used: Staining, CCK-8 Assay, Negative Control

    Graphical summary of this study. TfR: transferrin receptor, DMT1: divalent metal transporter 1, IRP2: Iron regulatory protein 2.
    Figure Legend Snippet: Graphical summary of this study. TfR: transferrin receptor, DMT1: divalent metal transporter 1, IRP2: Iron regulatory protein 2.

    Techniques Used:



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    Transferrin Receptor (Tfr) Antibody Mouse Monoclonal, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti-transferrin receptor mouse monoclonal antibody transr  (Thermo Fisher)
    90
    Thermo Fisher anti-transferrin receptor mouse monoclonal antibody transr

    Anti Transferrin Receptor Mouse Monoclonal Antibody Transr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti-transferrin receptor mouse monoclonal antibody transr/product/Thermo Fisher
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    ATP13A2 KD disrupts intracellular iron homeostasis in α-Syn-SH cells. We confirmed expression level of iron related genes for analysis of the effect of ATP13A2 on iron homeostasis. ( A – C ) mRNA expression of TfR ( A ), DMT1 ( B ), FPN ( C ) analyzed by qRT-PCR in α-Syn-SH cells with ATP13A2 KD. Data were normalized to the GAPDH level ( n = 4, biological replicates, TfR : NC = 1.0, siATP = 1.423, DMT1 : NC = 1.0, siATP = 1.362, FPN : NC = 1.0, siATP = 0.835). ( D ) Protein expression of TfR, DMT1, FPN, and IRP2 detected by Western blot in α-Syn-SH cells with ATP13A2 KD. (E–H) Quantification of ( D ). Data were normalized to β-actin levels ( n = 3, biological replicates, TfR: NC = 1.0, siATP = 1.648, DMT1: NC = 1.0, siATP = 0.971, FPN: NC = 1.0, siATP = 0.921, IRP2: NC = 1.0, siATP = 0.927). Each value represents the mean ± SEM. Student’s t-test was used to test the significance of differences (n.s. means not significant. * p < 0.05, ** p < 0.01, *** p < 0.001). siATP: siRNA targeting ATP13A2, NC: siRNA of negative control, TfR: Transferrin receptor, DMT1: Divalent Metal Transporter 1, FPN: Ferroportin, IRP2: Iron Regulatory protein 2.

    Journal: Scientific Reports

    Article Title: Disruption of intracellular iron homeostasis through mitochondrial dysfunction associated with suppression of ATP 13A2 expression

    doi: 10.1038/s41598-026-35368-x

    Figure Lengend Snippet: ATP13A2 KD disrupts intracellular iron homeostasis in α-Syn-SH cells. We confirmed expression level of iron related genes for analysis of the effect of ATP13A2 on iron homeostasis. ( A – C ) mRNA expression of TfR ( A ), DMT1 ( B ), FPN ( C ) analyzed by qRT-PCR in α-Syn-SH cells with ATP13A2 KD. Data were normalized to the GAPDH level ( n = 4, biological replicates, TfR : NC = 1.0, siATP = 1.423, DMT1 : NC = 1.0, siATP = 1.362, FPN : NC = 1.0, siATP = 0.835). ( D ) Protein expression of TfR, DMT1, FPN, and IRP2 detected by Western blot in α-Syn-SH cells with ATP13A2 KD. (E–H) Quantification of ( D ). Data were normalized to β-actin levels ( n = 3, biological replicates, TfR: NC = 1.0, siATP = 1.648, DMT1: NC = 1.0, siATP = 0.971, FPN: NC = 1.0, siATP = 0.921, IRP2: NC = 1.0, siATP = 0.927). Each value represents the mean ± SEM. Student’s t-test was used to test the significance of differences (n.s. means not significant. * p < 0.05, ** p < 0.01, *** p < 0.001). siATP: siRNA targeting ATP13A2, NC: siRNA of negative control, TfR: Transferrin receptor, DMT1: Divalent Metal Transporter 1, FPN: Ferroportin, IRP2: Iron Regulatory protein 2.

    Article Snippet: The transferred membrane was incubated in 5% skim milk (Nakarai Tesque) or Blocking One (Nakarai Tesque) at room temperature for 60 min. After blocking, the membrane was incubated with the following primary antibodies: the mouse monoclonal antibody transferrin receptor (1:500, Invitrogen), and β-actin (1:2000, Santa Cruz Biotechnology); rabbit polyclonal antibodies: ATP13A2 C-terminal region (1:1000, Sigma-Aldrich), LC3 (1:1000, MBL), SQSTM1/p62 (1:1000, Cell Signaling), IRP2 (1:1000, Novus Biologicals), ferritin and DMT1 (1:1000, Abcam) α-Synuclein (1:2000, Abcam) dissolved in 5% skim milk or Reagent A of Immuno-enhancer (FUJIFILM) at 4 °C overnight.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Negative Control

    Protective effect of ATP13A2 KD in α-Syn-SH cells by inhibiting iron influx. Apo Transferrin or Glibenclamide (GBD) were administered for 24 h after ATP13A2 KD in α-Syn-SH cells. ( A ) Atomic absorption spectrometry for evaluation of total intracellular iron level in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD. Quantification of iron content per 1.0 × 10 5 cells ( n = 3, biological replicates, NC = 1.579, NC apo-Tf = 1.645, siATP = 2.671, siATP apo-Tf = 1.984). ( B ) Staining of RhoNox-4 for evaluation of intracellular Fe 2+ level in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD (Red: Rho-Nox4, Blue: Hoechst) (Scale bar, 10 μm). ( C ) Quantification of (B) ( n = 5, biological replicates, Cell number of NC for each biological replicate: 156, 89, 93, 94, 116, Cell number of NC apo-Tf for each biological replicate: 146, 101, 117, 91, 76, Cell number of siATP for each biological replicate: 90, 134, 94, 108, 126, Cell number of siATP apo-Tf for each biological replicate: 135, 103, 68, 120, 112, NC = 1.0, NC apo-Tf = 1.069, siATP = 1.510, siATP apo-Tf = 1.121). ( D ) MitoSOX dye for detection of mitochondrial ROS in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD (Red: MitoSOX, Blue: Hoechst) (Scale bar, 20 μm). ( E ) Quantification of (D) ( n = 4, biological replicates, Cell number of NC for each biological replicate: 203, 180, 99, 163, Cell number of NC apo-Tf for each biological replicate: 236, 223, 208, 199, Cell number of siATP for each biological replicate: 187, 188, 175, 145, Cell number of siATP for each biological replicate: 187, 188, 175, 145, Cell number of siATP apo-Tf for each biological replicate: 162, 181, 173, 166, NC = 1.0, NC apo-Tf = 1.013, siATP = 2.729, siATP apo-Tf = 1.552). ( F ) Cell viability was measured by CCK-8 assay in α-Syn-SH cells treated with apo Transferrin for 24 h (0.01, 0.10, 0.50, or 1.00 mg/mL) after ATP13A2 KD ( n = 4, biological replicates, NC = 100, siATP = 78.34, siATP apo-Tf 0.01 mg/mL = 91.84, siATP apo-Tf 0.10 mg/mL = 102.3, siATP apo-Tf 0.50 mg/mL = 105.0, siATP apo-Tf 1.0 mg/mL = 101.8). ( G ) Atomic absorption spectrometry in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD. Quantification of iron content per 1.0 × 10 5 cells ( n = 3, biological replicates, NC = 1.276, NC GBD = 1.130, siATP = 1.882, siATP GBD = 1.268). ( H ) Staining of RhoNox-4 in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD (Red: Rho-Nox4, Blue: Hoechst) (Scale bar, 10 μm). ( I ) Quantification of (H) ( n = 5, biological replicates, Cell number of NC for each biological replicate: 73, 52, 60, 77, 54, Cell number of NC GBD for each biological replicate: 62, 86, 99, 96, 94, Cell number of siATP for each biological replicate: 123, 65, 80, 115, 97, Cell number of siATP GBD for each biological replicate: 82, 64, 81, 127, 95, NC = 1.0, NC GBD = 1.073, siATP = 1.849, siATP GBD = 1.126). ( J ) MitoSOX dye in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD (Red: MitoSOX, Blue: Hoechst) (Scale bar, 20 μm). ( K ) Quantification of (J) ( n = 4, biological replicates, Cell number of NC for each biological replicate: 121, 119, 153,128, Cell number of NC GBD for each biological replicate: 115, 158, 125, 139, Cell number of siATP for each biological replicate: 106, 109, 83, 99, Cell number of siATP GBD for each biological replicate: 119, 122, 128, 97, NC = 1.0, NC GBD = 0.904, siATP = 2.325, siATP GBD = 1.839). ( L ) Cell viability was measured by CCK-8 assay in α-Syn-SH cells treated with Glibenclamide for 24 h (3.0, 10.0, or 30.0 µM) after ATP13A2 KD ( n = 6, biological replicates, NC = 100, NC GBD 10 µM = 118.1, siATP = 73.66, siATP GBD 3 µM = 87.66, siATP GBD 10 µM = 94.94, siATP GBD 30 µM = 95.94). Each value represents the mean ± SEM. An ANOVA, followed by the Bonferroni/Dunn post-hoc test was used to test the significance of differences (n.s. not significant. * p < 0.05, ** p < 0.01, *** p < 0.001). siATP: siRNA targeting ATP13A2, NC: siRNA of negative control.

    Journal: Scientific Reports

    Article Title: Disruption of intracellular iron homeostasis through mitochondrial dysfunction associated with suppression of ATP 13A2 expression

    doi: 10.1038/s41598-026-35368-x

    Figure Lengend Snippet: Protective effect of ATP13A2 KD in α-Syn-SH cells by inhibiting iron influx. Apo Transferrin or Glibenclamide (GBD) were administered for 24 h after ATP13A2 KD in α-Syn-SH cells. ( A ) Atomic absorption spectrometry for evaluation of total intracellular iron level in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD. Quantification of iron content per 1.0 × 10 5 cells ( n = 3, biological replicates, NC = 1.579, NC apo-Tf = 1.645, siATP = 2.671, siATP apo-Tf = 1.984). ( B ) Staining of RhoNox-4 for evaluation of intracellular Fe 2+ level in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD (Red: Rho-Nox4, Blue: Hoechst) (Scale bar, 10 μm). ( C ) Quantification of (B) ( n = 5, biological replicates, Cell number of NC for each biological replicate: 156, 89, 93, 94, 116, Cell number of NC apo-Tf for each biological replicate: 146, 101, 117, 91, 76, Cell number of siATP for each biological replicate: 90, 134, 94, 108, 126, Cell number of siATP apo-Tf for each biological replicate: 135, 103, 68, 120, 112, NC = 1.0, NC apo-Tf = 1.069, siATP = 1.510, siATP apo-Tf = 1.121). ( D ) MitoSOX dye for detection of mitochondrial ROS in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD (Red: MitoSOX, Blue: Hoechst) (Scale bar, 20 μm). ( E ) Quantification of (D) ( n = 4, biological replicates, Cell number of NC for each biological replicate: 203, 180, 99, 163, Cell number of NC apo-Tf for each biological replicate: 236, 223, 208, 199, Cell number of siATP for each biological replicate: 187, 188, 175, 145, Cell number of siATP for each biological replicate: 187, 188, 175, 145, Cell number of siATP apo-Tf for each biological replicate: 162, 181, 173, 166, NC = 1.0, NC apo-Tf = 1.013, siATP = 2.729, siATP apo-Tf = 1.552). ( F ) Cell viability was measured by CCK-8 assay in α-Syn-SH cells treated with apo Transferrin for 24 h (0.01, 0.10, 0.50, or 1.00 mg/mL) after ATP13A2 KD ( n = 4, biological replicates, NC = 100, siATP = 78.34, siATP apo-Tf 0.01 mg/mL = 91.84, siATP apo-Tf 0.10 mg/mL = 102.3, siATP apo-Tf 0.50 mg/mL = 105.0, siATP apo-Tf 1.0 mg/mL = 101.8). ( G ) Atomic absorption spectrometry in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD. Quantification of iron content per 1.0 × 10 5 cells ( n = 3, biological replicates, NC = 1.276, NC GBD = 1.130, siATP = 1.882, siATP GBD = 1.268). ( H ) Staining of RhoNox-4 in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD (Red: Rho-Nox4, Blue: Hoechst) (Scale bar, 10 μm). ( I ) Quantification of (H) ( n = 5, biological replicates, Cell number of NC for each biological replicate: 73, 52, 60, 77, 54, Cell number of NC GBD for each biological replicate: 62, 86, 99, 96, 94, Cell number of siATP for each biological replicate: 123, 65, 80, 115, 97, Cell number of siATP GBD for each biological replicate: 82, 64, 81, 127, 95, NC = 1.0, NC GBD = 1.073, siATP = 1.849, siATP GBD = 1.126). ( J ) MitoSOX dye in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD (Red: MitoSOX, Blue: Hoechst) (Scale bar, 20 μm). ( K ) Quantification of (J) ( n = 4, biological replicates, Cell number of NC for each biological replicate: 121, 119, 153,128, Cell number of NC GBD for each biological replicate: 115, 158, 125, 139, Cell number of siATP for each biological replicate: 106, 109, 83, 99, Cell number of siATP GBD for each biological replicate: 119, 122, 128, 97, NC = 1.0, NC GBD = 0.904, siATP = 2.325, siATP GBD = 1.839). ( L ) Cell viability was measured by CCK-8 assay in α-Syn-SH cells treated with Glibenclamide for 24 h (3.0, 10.0, or 30.0 µM) after ATP13A2 KD ( n = 6, biological replicates, NC = 100, NC GBD 10 µM = 118.1, siATP = 73.66, siATP GBD 3 µM = 87.66, siATP GBD 10 µM = 94.94, siATP GBD 30 µM = 95.94). Each value represents the mean ± SEM. An ANOVA, followed by the Bonferroni/Dunn post-hoc test was used to test the significance of differences (n.s. not significant. * p < 0.05, ** p < 0.01, *** p < 0.001). siATP: siRNA targeting ATP13A2, NC: siRNA of negative control.

    Article Snippet: The transferred membrane was incubated in 5% skim milk (Nakarai Tesque) or Blocking One (Nakarai Tesque) at room temperature for 60 min. After blocking, the membrane was incubated with the following primary antibodies: the mouse monoclonal antibody transferrin receptor (1:500, Invitrogen), and β-actin (1:2000, Santa Cruz Biotechnology); rabbit polyclonal antibodies: ATP13A2 C-terminal region (1:1000, Sigma-Aldrich), LC3 (1:1000, MBL), SQSTM1/p62 (1:1000, Cell Signaling), IRP2 (1:1000, Novus Biologicals), ferritin and DMT1 (1:1000, Abcam) α-Synuclein (1:2000, Abcam) dissolved in 5% skim milk or Reagent A of Immuno-enhancer (FUJIFILM) at 4 °C overnight.

    Techniques: Staining, CCK-8 Assay, Negative Control

    Graphical summary of this study. TfR: transferrin receptor, DMT1: divalent metal transporter 1, IRP2: Iron regulatory protein 2.

    Journal: Scientific Reports

    Article Title: Disruption of intracellular iron homeostasis through mitochondrial dysfunction associated with suppression of ATP 13A2 expression

    doi: 10.1038/s41598-026-35368-x

    Figure Lengend Snippet: Graphical summary of this study. TfR: transferrin receptor, DMT1: divalent metal transporter 1, IRP2: Iron regulatory protein 2.

    Article Snippet: The transferred membrane was incubated in 5% skim milk (Nakarai Tesque) or Blocking One (Nakarai Tesque) at room temperature for 60 min. After blocking, the membrane was incubated with the following primary antibodies: the mouse monoclonal antibody transferrin receptor (1:500, Invitrogen), and β-actin (1:2000, Santa Cruz Biotechnology); rabbit polyclonal antibodies: ATP13A2 C-terminal region (1:1000, Sigma-Aldrich), LC3 (1:1000, MBL), SQSTM1/p62 (1:1000, Cell Signaling), IRP2 (1:1000, Novus Biologicals), ferritin and DMT1 (1:1000, Abcam) α-Synuclein (1:2000, Abcam) dissolved in 5% skim milk or Reagent A of Immuno-enhancer (FUJIFILM) at 4 °C overnight.

    Techniques:

    a Cultured human SHY-5Y neuroblastoma cells were transduced with recombinant lentiviruses leading to either REST inhibition (short hairpin RNA; sh4) or overexpression (human REST cDNA: hREST ), as previously described . Expression of γ-secretase components was assessed by qRT-PCR. b REST lx/lx MEF cells were transduced with Cre recombinase (generating REST-KO lines 1–3) or a control vector without Cre (lines WT 1–3). The REST floxed ( REST lx ) and Cre-recombined REST ( REST rec ) alleles were detected by PCR. No REST lx band was detected in REST-KO cells, suggesting complete Cre -mediated recombination. c qRT-PCR using two sets of primers spanning the transcript shows loss of REST mRNA in REST-KO MEFs. d Immunolabeling with anti-REST antibody (REST14 ; white) shows loss of REST expression in REST-KO MEFs. Nuclei are labeled with DAPI (blue). Scale bar, 40 μm. e Western blot analysis of γ-secretase components in WT and REST-KO MEFs. The transferrin receptor (TfR) served as loading control. f Quantification of protein levels normalized to TfR shown as percentage expression in REST-KO relative to WT cells (interrupted line: 100%). For PS1, similar results were seen with antibodies against the N-terminus (NTF; antibody 231f) or C-terminus (CTF; antibody EP2000Y). g REST-KO MEFs show elevated γ-secretase enzymatic activity. Solubilized membranes were incubated with Met-C99-FLAG in the presence or absence of a γ-secretase inhibitor (+I), and levels of AICD-FLAG were determined by Western blotting using TfR as a loading control. h Quantification of γ-secretase activity in membrane preparations from WT and REST-KO cells. AICD-FLAG levels were normalized to TfR, and shown as percentage expression in REST-KO relative to WT cells (interrupted line: 100%). Loss of REST in REST-KO MEFs leads to elevated Aβ40 ( i ) and Aβ42 ( j ) levels following transfection of hAPP WT or hAPP Swe . Lentiviral transduction of human REST cDNA (hREST) suppresses Aβ production. Individual values and the mean ± S.E.M are shown for n = 6 ( a ) or n = 3 ( c , f , h , i , j ) independent experiments. P -values are derived from two-tailed unpaired t -tests ( a , c , f , h ) or one-way ANOVA with Tukey’s post-hoc test ( i , j ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer’s disease

    doi: 10.1038/s41467-023-42704-6

    Figure Lengend Snippet: a Cultured human SHY-5Y neuroblastoma cells were transduced with recombinant lentiviruses leading to either REST inhibition (short hairpin RNA; sh4) or overexpression (human REST cDNA: hREST ), as previously described . Expression of γ-secretase components was assessed by qRT-PCR. b REST lx/lx MEF cells were transduced with Cre recombinase (generating REST-KO lines 1–3) or a control vector without Cre (lines WT 1–3). The REST floxed ( REST lx ) and Cre-recombined REST ( REST rec ) alleles were detected by PCR. No REST lx band was detected in REST-KO cells, suggesting complete Cre -mediated recombination. c qRT-PCR using two sets of primers spanning the transcript shows loss of REST mRNA in REST-KO MEFs. d Immunolabeling with anti-REST antibody (REST14 ; white) shows loss of REST expression in REST-KO MEFs. Nuclei are labeled with DAPI (blue). Scale bar, 40 μm. e Western blot analysis of γ-secretase components in WT and REST-KO MEFs. The transferrin receptor (TfR) served as loading control. f Quantification of protein levels normalized to TfR shown as percentage expression in REST-KO relative to WT cells (interrupted line: 100%). For PS1, similar results were seen with antibodies against the N-terminus (NTF; antibody 231f) or C-terminus (CTF; antibody EP2000Y). g REST-KO MEFs show elevated γ-secretase enzymatic activity. Solubilized membranes were incubated with Met-C99-FLAG in the presence or absence of a γ-secretase inhibitor (+I), and levels of AICD-FLAG were determined by Western blotting using TfR as a loading control. h Quantification of γ-secretase activity in membrane preparations from WT and REST-KO cells. AICD-FLAG levels were normalized to TfR, and shown as percentage expression in REST-KO relative to WT cells (interrupted line: 100%). Loss of REST in REST-KO MEFs leads to elevated Aβ40 ( i ) and Aβ42 ( j ) levels following transfection of hAPP WT or hAPP Swe . Lentiviral transduction of human REST cDNA (hREST) suppresses Aβ production. Individual values and the mean ± S.E.M are shown for n = 6 ( a ) or n = 3 ( c , f , h , i , j ) independent experiments. P -values are derived from two-tailed unpaired t -tests ( a , c , f , h ) or one-way ANOVA with Tukey’s post-hoc test ( i , j ). Source data are provided as a Source Data file.

    Article Snippet: Additional primary antibodies were as follows: anti-human Aβ rabbit monoclonal IgG antibody (Cell Signaling, Cat. No. 8243); anti-human APP mouse monoclonal IgG antibody (clone 6E10; Covance, Catalog No. SIG-39320); anti-actin mouse monoclonal IgG antibody (clone ACTN05 (C4); ThermoFisher Scientific, Catalog No. MA5-11869); anti-NeuN mouse monoclonal IgG antibody (clone A60, Millipore, MAB377); anti-MAP2 goat polyclonal IgG antibody (PhosphoSolutions, Catalog. No. 1099-MAP2); anti-CDK5 mouse monoclonal IgG antibody (clone 4E4; Novus Bio, Catalog No. NBP2-37602); anti-GSK3β mouse monoclonal IgG antibody (clone D5C5Z; Novus Bio, catalog No. NBP1-47470S); anti-PS1 C-terminal (CTF) rabbit monoclonal IgG antibody (clone EP2000Y; Abcam, Catalog No. ab76083); anti-PS1 N-terminal (NTF) rabbit polyclonal IgG antibody (231-f; made in the Yankner lab); anti-Nicastrin mouse monoclonal IgG antibody (clone 9C3; Biolegend, Catalog No. 852301); anti-Nicastrin rabbit polyclonal IgG antibody (Sigma Millipore, Catalog No. N1660); anti-PEN2 rabbit polyclonal IgG antibody (ProScience, Catalog No.3981); anti-PEN2 rabbit monoclonal IgG antibody (clone EPR9200; Abcam, Catalog No. ab154830); anti-PEN2 rabbit polyclonal IgG (ProScience, Catalog No. 3981); anti-Transferrin receptor mouse monoclonal IgG antibody (clone H68.4; ThermoFisher Scientific, Catalog No. 13-6800); anti-BiP/GRP78 mouse monoclonal IgG (clone C38; ThermoFisher Scientific, clone C38, Catalog No. 14-9768-82); anti-β-catenin goat polyclonal IgG (R&D Systems, Catalog No. AF1329); non-specific rabbit IgG antibody (Sigma Millipore, Catalog No. 17-641); and anti-FLAG mouse monoclonal IgG1 antibody (clone M2; Sigma Millipore, Catalog No. F3165).

    Techniques: Cell Culture, Transduction, Recombinant, Inhibition, shRNA, Over Expression, Expressing, Quantitative RT-PCR, Control, Plasmid Preparation, Immunolabeling, Labeling, Western Blot, Activity Assay, Incubation, Membrane, Transfection, Derivative Assay, Two Tailed Test

    Journal: eLife

    Article Title: The PMA phorbol ester tumor promoter increases canonical Wnt signaling via macropinocytosis

    doi: 10.7554/eLife.89141

    Figure Lengend Snippet:

    Article Snippet: Antibody , Transferrin receptor (TfR) antibody mouse monoclonal , Thermo Fisher , Cat# 13-6800 , IF (1:100).

    Techniques: Recombinant, Dominant Negative Mutation, Plasmid Preparation, Membrane, Luciferase, Expressing, Reporter Assay, Software, Imaging, Microinjection, Inverted Microscopy