Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Both PCV2 and ORF5 reduced the expression of GPNMB in PAMs. (A) PCR detection of PCV2 nucleic acid in PAM cells with PCV2 infection at a MOI of 1.0 at 24 and 48 h post-infection. Marker. DNA Marker DL1000. (B) Real-time qRT-PCR analysis of GPNMB mRNA expression in PAM cells infected with PCV2 a MOI of 1 at indicated time points. (C) Immunoblot analysis of GPNMB protein expression in PAM cells infected with PCV2 a MOI of 1 at indicated time points. (D) Immunoblot analysis of GFP protein expression in PAM cells transfected with GFP-C1 or GFP ORF5 construct at 48 h post-transfection. (E) Real-time qRT-PCR analysis of GPNMB mRNA expression in PAM cells transfected with GFP-C1 or GFP ORF5 construct at indicated time points. (F) Immunoblot analysis of GPNMB protein expression in PAM cells transfected with GFP-C1 or GFP ORF5 construct at indicated time points. Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in (B) and (E) . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05; ∗∗∗ p < 0.001.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Expressing, Infection, Marker, Quantitative RT-PCR, Western Blot, Transfection, Construct

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Interaction of ORF5 with GPNMB. (A) Exogenous co-IP analysis of ORF5 and GPNMB in PAMs. Cells were co-transfected with plasmids GFP-ORF5 and Flag-GPNMB. PAMs co-transfected with GFP-C1 and Flag-GPNMB were used as negative controls. A quarter of the cell extract was subjected to the input assay to assess β-actin, Flag-fusion and GFP-fusion protein levels. The rest of the extract was subjected to IP assay. Western blot detected proteins with a mouse anti-GFP mAb and a mouse anti-Flag pAb. (B) ORF5 protein co-localizes with GPNMB. HEK-293T cells were co-transfected with pGPNMB-Red and pORF5-GFP, pEGFP-C1 and pDsRed-N1 were used as control. Cells were fixed and stained with DAPI (blue) at 48 h post-transfection. Scale bar = 10 μm. (C) Endogenous co-IP analysis of ORF5 and GPNMB in PAMs. Cells were transfected with plasmid GFP-ORF5-Flag and GFP-C1-transfected PAMs were used as negative controls. The input assay was performed using a quarter of the cell extract to assess β-actin, Flag fusion protein and GPNMB levels. (D) GST-ORF5 pull-down assay. The GST and GST-ORF5 proteins expressed in Escherichia coli Rosetta (DE3) cells were immobilized on a glutathione agarose resin, followed by incubation of the resin with the cell lysates containing GPNMB-Flag protein.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, Staining, Plasmid Preparation, Pull Down Assay, Incubation

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Stable overexpression of GPNMB inhibits PCV2 replication and ORF5 expression. (A) Real-time qRT-PCR analysis of GPNMB mRNA expression in PK-15 cells with stable GPNMB overexpression. (B) Immunoblot analysis of GPNMB protein in PK-15 cells with stable GPNMB overexpression. The right panel is the GPNMB protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (C,D) Real-time qRT-PCR analysis of PCV2 viral RNA (C) and ORF5 mRNA (D) expression in PK-15 cells with stable GPNMB overexpression. Different cell lines were infected with PCV2 at a MOI of 0.1. PCV2 viral RNA level was measured 24 h or 48 h post-infection. Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A , C , and D . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Infection

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Transient overexpression of Red-fused GPNMB inhibits PCV2 replication and ORF5 expression. (A) Real-time qRT-PCR analysis of GPNMB mRNA expression in PK-15 cells transfected with pDsRed-GPNMB or PdsRed-N1 at 24 h post-transfection. (B) Immunoblot analysis of GPNMB protein in PK-15 cells transfected with pDsRed-GPNMB or PdsRed-N1 at 24 h post-transfection. The right panel is the GPNMB protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (C,D) Real-time qRT-PCR analysis of PCV2 viral RNA (C) and ORF5 mRNA (D) expression in PK-15 cells transfected with pDsRed-GPNMB or PdsRed-N1. Different cells were infected with PCV2 at a MOI of 0.1 at 24 h post-transfection. RNA expression level was measured at 24 h post-infection. Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A , C , and D . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Infection, RNA Expression

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Knockdown of GPNMB increases PCV2 replication and ORF5 expression. (A) Real-time qRT-PCR analysis of GPNMB mRNA expression in stable GPNMB knockdown PK-15 cells. PK-15 cells transduced with GPNMB-knockdown lentivirus (shGPNMB-1, 2, 3) or random sequence control (shN). (B) Immunoblot analysis of GPNMB protein in stable GPNMB knockdown PK-15 cells. The right panel is the GPNMB protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (C,D) Real-time qRT-PCR analysis of PCV2 viral RNA (C) and ORF5 mRNA (D) expression in stable GPNMB knockdown. Different cells were infected with PCV2 at a MOI of 0.1. RNA expression level was measured at 24 h or 48 h post-infection. Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A , C , and D . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Expressing, Quantitative RT-PCR, Transduction, Sequencing, Western Blot, Infection, RNA Expression

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Overexpression of GPNMB promotes cell cycle progression. (A) Real-time qRT-PCR analysis of Cyclin A mRNA expression in PAM cells transfected with pEGFP-C1 or pEGFP-ORF5 construct at 24 h or 48 h post-transfection. (B) Immunoblot analysis of Cyclin A protein expression in PAM cells transfected with pEGFP-C1 or pEGFP-ORF5 construct at 24 h or 48 h post-transfection. (C,D) Real-time qRT-PCR analysis of GPNMB (C) and Cyclin A (D) mRNA expression in PAMs with stable GPNMB overexpression. (E) Immunoblot analysis of GPNMB and Cyclin A protein levels in PAM cells with stable GPNMB overexpression. The middle panel is the GPNMB protein level and the right panel is the Cyclin A protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (F) Histograms from flow cytometry data for propidium iodide (PI) staining for the mock cells (Mock), control lentivirus transduced cells (Lenti) and GPNMB overexpression lentivirus transduced cells (Lenti-GPNMB). Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A and B . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Construct, Western Blot, Flow Cytometry, Staining

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Knockdown of GPNMB inhibits cell cycle progression. (A,B) Real-time qRT-PCR analysis of GPNMB mRNA (A) and Cyclin A mRNA (B) expression in PAMs cell lines with GPNMB knockdown. (C) Immunoblot analysis of GPNMB and Cyclin A protein levels in PAM cells with lines with GPNMB knockdown. The middle panel is the GPNMB protein level and the right panel is the Cyclin A protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (D) Histograms from flow cytometry data for propidium iodide (PI) staining for the mock cells (Mock), control lentivirus transduced cells (shN) and GPNMB knockdown lentivirus transduced cells (shGPNMB-3). Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A and B . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Slingshot homolog-1-mediated Nrf2 sequestration tips the balance from neuroprotection to neurodegeneration in Alzheimer's disease.

doi: 10.1073/pnas.2217128120

Figure Lengend Snippet: Fig. 1. SSH1 inhibits Nrf2/ARE target gene expression independent of SSH1 phosphatase activity. (A1) Schematic of the Nrf2 reporter construct pREP-8xARE- GFP-SV40-BFP. (A2) Schematic of SSH1 and SSH1-CS proteins showing the catalytic domain (CAT) and binding sites for cofilin and p62. (B) Representative images of HT22 cells coexpressing the Nrf2 reporter (green and blue), with vector control, myc-Nrf2, and/or Flag-SSH1 (red). (C) Quantification of Nrf2 reporter [one- way ANOVA, F (2, 218) = 18.62, P < 0.0001, post hoc Dunnett, ****P < 0.0001, **P = 0.0076, n = 15 to 20 images/condition/experiment from three experiments]. (D) Representative images of HT22 cells coexpressing the Nrf2 reporter (green and blue) and vector or Flag-SSH1 (red), ± 200 μM H2O2 (2 h). (E) Quantification of Nrf2 reporter [one-way ANOVA, F (2, 519) = 124.7, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 15 to 20 images/condition/experiment from four experiments]. (F) Representative images of HT22 cells coexpressing the Nrf2 reporter (green and blue), vector, Flag-SSH1, or Flag-SSH1CS (red), ± 250 μM H2O2 (3 h). (G) Quantification of Nrf2 reporter [one-way ANOVA, F (3, 308) = 61.03, P < 0.0001, post hoc Dunnett, ****P < 0.0001, ns = not significant, n = 8 to 12 images/ condition/experiment from four experiments]. (H) Representative images of HT22 cells cotransfected with the Nrf2 reporter (green and blue) and control siRNA or SSH1 siRNA, stained for SSH1 (red), ± 250 μM H2O2 (3 h). (I) Quantification of Nrf2 reporter [one-way ANOVA, F (3, 855) = 41.31, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 15 to 20 images/condition/experiment from four experiments]. (J) Representative immunoblots from lysates of HEK293T cells expressing vector or Flag-SSH1 ± 15 µM NaAsO2 (18 h). (K) Quantification of HMOX1 and NQO1 proteins [one-way ANOVA; HMOX1: F (2, 21) = 310.2, P < 0.0001; NQO1: F (2, 21) = 34.82, P < 0.0001; post hoc Dunnett, ****P < 0.0001, ***P < 0.001, *P < 0.05. n = 8 samples/condition]. (L) Representative immunoblots from lysates of HEK293T cells coexpressing vector or myc-Nrf2 plus vector, Flag-SSH1, or Flag-SSH1-CS. (M) Quantification of HMOX1 protein [one-way ANOVA, F (2, 6) = 32.61, P = 0.0006, post hoc Dunnett, ***P = 0.006, **P = 0.0022, ns = not significant, n = 3 samples/condition]. (N) Representative immunoblots from lysates of HEK293T cells transfected with control or SSH1 siRNA ± 15 µM NaAsO2 (18 h). (O) Quantification of HMOX1 protein [one-way ANOVA, F (2, 15) = 185.4, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 6 samples/condition]. (P) Quantification of NQO1 protein [one-way ANOVA, F (2, 14) = 34.49, P < 0.0001, post hoc Dunnett, ****P < 0.0001, ns = not significant, n = 6 samples/condition].

Article Snippet: The following antibodies were used to probe target proteins: rabbit polyclonal anti- SSH1(ECM Biosciences, SP1711); mouse monoclonal anti- Nrf2 (3G7)(Novus Biologicals, AF4000); rabbit monoclonal anti- Keap1 (D6B12) (Cell Signaling Technologies, 8,047); rabbit monoclonal anti- SQSTM1/p62 (D10E10) (Cell Signaling Technologies, 7,695); mouse monoclonal anti- FLAG (M2) (Sigma- Aldrich, F3165); rabbit monoclonal anti- Nrf2 (D1Z9C) (Cell Signaling Technologies, 12,721); mouse monoclonal anti- Keap1 (1B4) (Abcam, ab119403); mouse monoclonal antiSSH1 (1A5C8) (Santa Cruz Biotech, sc- 517226); Alexa Fluor 594 goat anti- rabbit IgG (Invitrogen, A11037); Alexa Fluor 594 goat anti- mouse IgG (Invitrogen, A11032); and Alexa Fluor 488 goat anti- rabbit IgG (Invitrogen, A11034).

Techniques: Targeted Gene Expression, Activity Assay, Construct, Binding Assay, Plasmid Preparation, Control, Staining, Western Blot, Expressing, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Slingshot homolog-1-mediated Nrf2 sequestration tips the balance from neuroprotection to neurodegeneration in Alzheimer's disease.

doi: 10.1073/pnas.2217128120

Figure Lengend Snippet: Fig. 3. AD and FTLD-tau brains exhibit excessive levels of inhibitory SSH1–Nrf2 and Keap1–Nrf2 interactions. (A) Representative images of HT22 cells transfected with GFP (green) and vector or Flag-SSH1, treated ± 8 µM NaAsO2 (14 h) and subjected to PLA for SSH1–Nrf2 (red). (B) Quantification of SSH1–Nrf2 PLA puncta area/cell [Brown-Forsythe and Welch ANOVA, F (3, 202.7) = 30.48, F (3, 183.2) = 62.13, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 10 to 15 images/ condition/experiment from four experiments]. (C) Representative images of human frontal gyrus sections from nondementia, AD, and FTLD-tau cases showing DAPI (blue) and PLA for SSH1–Nrf2 (red) and Keap1–Nrf2 (red). (D and E) Quantification of SSH1–Nrf2 PLA area in (D) control vs. AD (two-tailed t test, t = 6.329, df = 156, ****P < 0.0001, n = 8 to 10 images/case from 7 to 8 case/condition) and (E) control vs. FTLD-tau (two-tailed t test, t = 2.353, df = 157, *P = 0.0199, n = 8 to 10 images/case from eight cases/condition). (F and G) Quantification of Keap1–Nrf2 PLA area in (F) control vs. AD (two-tailed t test, t = 3.371, df = 154, ***P = 0.0009; n = 8 to 10 images/case from 7 to 8 cases/condition) and (G) control vs. FTLD-tau (two-tailed t test, t = 4.752, df = 152; ****P < 0.0001; n = 8 to 10 images/ case from eight cases/condition).

Article Snippet: The following antibodies were used to probe target proteins: rabbit polyclonal anti- SSH1(ECM Biosciences, SP1711); mouse monoclonal anti- Nrf2 (3G7)(Novus Biologicals, AF4000); rabbit monoclonal anti- Keap1 (D6B12) (Cell Signaling Technologies, 8,047); rabbit monoclonal anti- SQSTM1/p62 (D10E10) (Cell Signaling Technologies, 7,695); mouse monoclonal anti- FLAG (M2) (Sigma- Aldrich, F3165); rabbit monoclonal anti- Nrf2 (D1Z9C) (Cell Signaling Technologies, 12,721); mouse monoclonal anti- Keap1 (1B4) (Abcam, ab119403); mouse monoclonal antiSSH1 (1A5C8) (Santa Cruz Biotech, sc- 517226); Alexa Fluor 594 goat anti- rabbit IgG (Invitrogen, A11037); Alexa Fluor 594 goat anti- mouse IgG (Invitrogen, A11032); and Alexa Fluor 488 goat anti- rabbit IgG (Invitrogen, A11034).

Techniques: Transfection, Plasmid Preparation, Control, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Slingshot homolog-1-mediated Nrf2 sequestration tips the balance from neuroprotection to neurodegeneration in Alzheimer's disease.

doi: 10.1073/pnas.2217128120

Figure Lengend Snippet: Fig. 4. Ssh1 elimination increases nuclear Nrf2, reduces oxidative injury, and alleviates AD pathology. (A) Representative images of the cortex from 7-mo-old WT, P301S, and P301S;Ssh1−/− mice stained for Nrf2 (green) and DAPI (blue). White boxes serially magnified to the right. (B) Quantification of nuclear/cytoplasmic Nrf2 intensity [one-way ANOVA, F (2, 134) = 13.06, P < 0.0001, post hoc Dunnett, ****P < 0.0001, **P = 0.0013, n = 10 to 12 images/mouse from four mice/genotype]. (C) Representative images of the cortex and hippocampus (CA3) stained for 8-OHdG (green) and DAPI (blue) from 7-mo-old WT, P301S, and P301S;Ssh1−/− mice. (D and E) Quantification of 8-OHdG intensity in the (D) cortex [one-way ANOVA, F (2, 134) = 37.79, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 10 to 13 images/mouse from four mice/genotype) and (E) hippocampus [one-way ANOVA, F (2, 70) = 17.85, P < 0.0001, post hoc Dunnett, ****P < 0.0001, ***P = 0.0003, n = 5 to 7 images/mouse from four mice/genotype]. (F) Representative images of the cortex from 8-mo-old WT, APP/PS1, and APP/PS1;Ssh1−/− mice stained for 8-OHdG (red) and DAPI (blue). (G) Quantification of 8-OHdG intensity [one-way ANOVA, F (2, 181) = 36.83, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 10 to 12 images/mouse from four mice/genotype]. (H) Representative images of silver staining of the cortex and hippocampus from 7-mo-old WT, P301S, and P301S;Ssh1−/− mice. Red arrows indicate silver-positive degenerating axons. (I and J) Quantification of silver-positive axons in the (I) cortex [one-way ANOVA, F (2, 21) = 14.5, P = 0.0001, post hoc Dunnett, ***P < 0.0005, n = 6 to 10 mice/genotype] and (J) hippocampus [one-way ANOVA, F (2, 21) = 7.545, P = 0.0034, post hoc Dunnett, **P < 0.0081, n = 6 to 10 mice/genotype]. (K) Representative images of the cortex and hippocampus (CA3) stained for pS199/202-tau (green) and DAPI (blue) from 7-mo-old P301S and P301S;Ssh1−/− mice. (L and M) Quantification of pS199/202-tau intensity in the (L) cortex (two-tailed t test, t = 13.85, df = 221, ****P < 0.0001, n = 20 to 30 images/mouse from four mice/genotype) and (M) hippocampus (two-tailed t–test, t = 8.147, df = 104, ****P < 0.0001, n = 13 to 18 images/mouse from four mice/genotype). (N) Representative images of the cortex and hippocampus stained for Aβ (green) and DAPI (blue) from 8-mo-old WT, APP/PS1, and APP/PS1;Ssh1−/− mice. (O) Quantification of Aβ intensity in the cortex (two-tailed t test, t = 2.784, df = 76, **P = 0.0068, n = 6 to 8 images/mouse from 4 to 6 mice/genotype).

Article Snippet: The following antibodies were used to probe target proteins: rabbit polyclonal anti- SSH1(ECM Biosciences, SP1711); mouse monoclonal anti- Nrf2 (3G7)(Novus Biologicals, AF4000); rabbit monoclonal anti- Keap1 (D6B12) (Cell Signaling Technologies, 8,047); rabbit monoclonal anti- SQSTM1/p62 (D10E10) (Cell Signaling Technologies, 7,695); mouse monoclonal anti- FLAG (M2) (Sigma- Aldrich, F3165); rabbit monoclonal anti- Nrf2 (D1Z9C) (Cell Signaling Technologies, 12,721); mouse monoclonal anti- Keap1 (1B4) (Abcam, ab119403); mouse monoclonal antiSSH1 (1A5C8) (Santa Cruz Biotech, sc- 517226); Alexa Fluor 594 goat anti- rabbit IgG (Invitrogen, A11037); Alexa Fluor 594 goat anti- mouse IgG (Invitrogen, A11032); and Alexa Fluor 488 goat anti- rabbit IgG (Invitrogen, A11034).

Techniques: Staining, Silver Staining, Two Tailed Test