Journal: PLoS ONE

Article Title: Ectodermal-Neural Cortex 1 Down-Regulates Nrf2 at the Translational Level

doi: 10.1371/journal.pone.0005492

Figure Lengend Snippet: A. MDA-MB-231 cells were transfected with an expression vector for HA-Nrf2, along with an empty vector or a plasmid for ENC1-Myc, Keap1-CBD, GAN1-Myc or Sarcosin-Myc. Transfected cells were lysed at 48 hr post-transfection and cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc, anti-CBD and anti-tubulin antibodies. B. An expression vector for HA-Nrf2 or HA-SMN gene was transfected into MDA-MB-231 cells, along with an empty vector or an expression vector for ENC1-Myc. Cells were harvested and immunoblot analysis was performed. C. An expression vector for ENC1-Myc was transfected into MDA-MB-231 cells. Cells were left untreated or treated with 50 µM tBHQ for 16 hr before cell lysis. Cell lysates were subjected to immunoblot analysis (left panel). The intensities of the Nrf2 bands were quantified by the ChemiDoc XRS gel documentation system and Quantity One software from Bio-Rad (right panel). D. MDA-MB-231 cells were transfected with different amounts of an ENC1-Myc expression vector, along with an expression vector for HA-Nrf2. Cell lysates were subjected to immunoblot analysis with anti-HA and anti-Derlin-1 antibodies.

Article Snippet: Antibodies against Nrf2, Myc, HA, β-Actin, Tubulin, Gal4 (Santa Cruz Biotechnology), and CBD (New England Biolabs) were purchased from commercial sources.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Lysis, Software

Journal: PLoS ONE

Article Title: Ectodermal-Neural Cortex 1 Down-Regulates Nrf2 at the Translational Level

doi: 10.1371/journal.pone.0005492

Figure Lengend Snippet: A. MDA-MB-231 cells transfected with plasmids for ARE-driven firefly luciferase, TK-renilla luciferase and the indicated genes were treated with 7.5 µM SF or 50 µM tBHQ for 16 hr prior to cell lysis. Firefly and renilla luciferase activities were measured. Means and standard deviations were calculated from three independent experiments. B. Small aliquots of the lysates were subjected to immunoblot analysis with anti-Nrf2 (left panel), anti-HA, anti-Myc, anti-CBD and anti-β-actin antibodies (right panel). C. An empty plasmid or a plasmid for either ENC1-Myc or Keap1 was transfected into MDA-MB-231 cells and cells were treated with tBHQ for 16 hr. At 48 hr post-transfection, the relative mRNA levels of Nrf2, NQO1, HO-1, ENC1 and Keap1 were determined by real-time PCR.

Article Snippet: Antibodies against Nrf2, Myc, HA, β-Actin, Tubulin, Gal4 (Santa Cruz Biotechnology), and CBD (New England Biolabs) were purchased from commercial sources.

Techniques: Transfection, Luciferase, Lysis, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Ectodermal-Neural Cortex 1 Down-Regulates Nrf2 at the Translational Level

doi: 10.1371/journal.pone.0005492

Figure Lengend Snippet: A. MDA-MB-231 cells were transfected with an empty vector or a vector for ENC1-Myc. Transfected cells were treated with proteasome inhibitors MG132 (M) (10 µM), clasto-lactacystin β-lactone (Lac) (10 µM) or epoxomicin (Ep) (1 µM), or lysosome inhibitors chloroquine (Ch) (50 µM) or ammonium chloride (Ac) (50 mM) for 4 hr before cells were lysed at 48 hr post-transfection. Cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc and anti-β-actin antibodies. B. MDA-MB-231 cells were transfected with HA-Nrf2 and with an empty vector or ENC1-Myc. Transfected cells were treated with proteasome and lysosome inhibitors as described above. Immunoblot analysis was performed. C. In vivo ubiquitination assay was performed in MDA-MB-231 cells transfected with plasmids for HA-Ub and Gal4-Neh2, along with either ENC1-Myc or Keap1-CBD. Transfected cells were treated with 10 µM MG132 for 4 hr prior to cell lysis. Cell lysates were denatured by heating and subjected to immunoprecipitation with anti-Gal4 antibodies. The precipitated protein complexes were subjected to immunoblot analysis with anti-HA antibodies for detecting ubiquitin-conjugated Gal4-Neh2 (top panel). Small aliquots of total cell lysates were immunoblotted with the indicated antibodies (bottom three panels).

Article Snippet: Antibodies against Nrf2, Myc, HA, β-Actin, Tubulin, Gal4 (Santa Cruz Biotechnology), and CBD (New England Biolabs) were purchased from commercial sources.

Techniques: Transfection, Plasmid Preparation, Western Blot, In Vivo, Ubiquitin Assay, Lysis, Immunoprecipitation

Journal: PLoS ONE

Article Title: Ectodermal-Neural Cortex 1 Down-Regulates Nrf2 at the Translational Level

doi: 10.1371/journal.pone.0005492

Figure Lengend Snippet: A. MDA-MB-231 cells were transfected with an expression vector for ENC1-Myc, along with an empty vector or an expression vector for Keap1-CBD. Cell lysates were subjected to chitin pull-down assay. Precipitated proteins were subjected to immunoblot analysis with anti-Myc and anti-CBD antibodies for detection of Keap1-CBD and ENC1-Myc (top panel). Small aliquots of total lysates were analyzed by immunoblot with the indicated antibodies (bottom panel). B. Cell lysates from MDA-MB-231 cells were immunoprecipitated with anti-Keap1 antibody. The precipitated protein complexes were subjected to immunoblot analysis with anti-Nrf2 and anti-ENC1 antibodies. IgG was included in the immunoprecipitation analysis as a negative control. C. Plasmids for full-length ENC1 (E-FL) and two ENC1 deletion mutants E320 and E570, Nrf2 and Luciferase (Luc) were used for in vitro transcription/translation to synthesize [ 35 S]-labeled proteins. The proteins were incubated with Keap1-His purified from E. Coli . Protein complexes containing Keap1 were pulled-down with nickel beads and resolved in SDS-PAGE and detected by autoradiography. Nrf2 and Luciferase were used as a positive and a negative control. 20% of [ 35 S]-labeled proteins were resolved by SDS-PAGE gel to show equivalent input of each protein (right panel). D. Keap1-/- and wild-type MEF cells were transfected with plasmids for HA-Nrf2 and ENC1-Myc. Cells were lysed at 48 hr post-transfection. Cell lysates were analyzed by immunoblot with anti-HA, anti-Myc and anti-β-actin. E. Cell lysates from MDA-MB-231 cells transfected with plasmids for Keap1-CBD, HA-Nrf2, and ENC1-Myc were used for pull-down assay with chitin beads Precipitated proteins were subjected to immunoblot analysis with anti-HA and anti-CBD antibodies (left figure, top two panels). Small aliquots of total lysates were analyzed by immunoblot with the indicated antibodies (left figure, bottom four panels). Nrf2 and β-Actin band intensities were quantified using Quantity One (BIO-RAD). The intensity of Nrf2 was normalized to that of β-actin (right graph).

Article Snippet: Antibodies against Nrf2, Myc, HA, β-Actin, Tubulin, Gal4 (Santa Cruz Biotechnology), and CBD (New England Biolabs) were purchased from commercial sources.

Techniques: Transfection, Expressing, Plasmid Preparation, Pull Down Assay, Western Blot, Immunoprecipitation, Negative Control, Luciferase, In Vitro, Labeling, Incubation, Purification, SDS Page, Autoradiography

Journal: PLoS ONE

Article Title: Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response

doi: 10.1371/journal.pone.0006588

Figure Lengend Snippet: (A) HEK293T cells were cotransfected with expression vectors for the indicated MAPKs, HA-tagged Nrf2, and CBD-tagged Keap1. Cells were harvested and lysed in RIPA buffer. Cell lysates were pulled-down with chitin beads and analyzed by immunoblot with anti-HA and anti-CBD antibodies. CBD: chitin binding domain; wt: wild-type; 5A: mutant with combined alanine substitution on all five phosphorylation sites. (B) NIH3T3 cells were transfected with expression vectors for the indicated Nrf2 protein and Keap1. The subcellular localization of Nrf2 was determined by indirect immunofluorescence analysis with anti-HA antibodies. (C) Quantification analysis of the immunofluorescence assay. At least 100 positively stained cells were examined by fluorescence microscopy. Percentages of cells in which Nrf2 was localized predominantly in the cytosol, the whole cell, or the nucleus were presented as a bar graph.

Article Snippet: Antibodies against the HA epitope (Santa Cruz), chitin-binding domain (CBD) (New England Biolab), α-tubulin (Santa Cruz), and phosphorylated serine/threonine followed by a proline (pS/TP) (Abcam) were purchased from commercial sources.

Techniques: Expressing, Western Blot, Binding Assay, Mutagenesis, Transfection, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: NPJ Genomic Medicine

Article Title: Novel homozygous nonsense mutation of MLIP and compensatory alternative splicing

doi: 10.1038/s41525-022-00307-y

Figure Lengend Snippet: a Volcano Plot highlighting the most differentially expressed genes in Z46 compared to controls following DESeq analysis. b RT-qPCR reveals mRNA expression levels for the targeted genes: MLIP 5–6 accounts specifically for transcripts containing the nonsense mutation, MLIP 9–10 covers all the known transcripts, and LMNA probe accounts for both lamin A and C. RPS29 is used for data normalization, error bars correspond to standard deviation (triplicata). c, d Western Blot analysis reveals the presence of four isoforms of MLIP, two of which can be quantified (27 kDa and 50 kDa). Both lamin types also appear for LMNA and were analyzed separately. GAPDH is used as the reference gene for normalization of the blot in both instances.

Article Snippet: Antibodies used in this report: MLIP rabbit-polyclonal (Invitrogen PA5–72759, 1:2000 dilution); LMNA rabbit-polyclonal (NEB 2026 S, 1:1000 dilution); GAPDH rabbit-monoclonal (NEB 5174 S, 1:1000 dilution); HRP anti-rabbit (Jackson ImmunoResearch 111–035–114, 1:10000 dilution).

Techniques: Quantitative RT-PCR, Expressing, Mutagenesis, Standard Deviation, Western Blot

Journal: The Journal of Cell Biology

Article Title: Sequential dynein effectors regulate axonal autophagosome motility in a maturation-dependent pathway

doi: 10.1083/jcb.202010179

Figure Lengend Snippet: Antibodies

Article Snippet: Anti-CBD , E80345 (New England Biolabs) , WB @ 1:800 , .

Techniques: Western Blot, Proximity Ligation Assay