Journal: Cells

Article Title: A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes

doi: 10.3390/cells3041131

Figure Lengend Snippet: Mutation of PML phosphorylation sites does not affect colocalization of PML-I and ICP0 during HSV-1 infection. HA-shPML cells transduced with FLAG-, CFP-tagged PML-I or each PML-I mutant were infected with HSV-1 at 2 PFU/cell. At 2 hpi, the cells were fixed and stained with antibodies against ICP0 and ICP4. PML is shown as red, ICP0 as green, and ICP4 as blue in the merged image. Scale bar = 10 μm.

Article Snippet: The transduced and infected cells were probed first with antibodies against ICP0 (H11060, Santa Cruz Biotechnology) and ICP4 (EastCoastBio) diluted in 5% rabbit serum in PBS and then probed with goat-anti-mouse IgG1 Dylight 594 and goat-anti-mouse IgG2b Dylight 488 as listed above for Sp100 and Daxx staining.

Techniques: Mutagenesis, Phospho-proteomics, Infection, Transduction, Staining

Journal: Cells

Article Title: A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes

doi: 10.3390/cells3041131

Figure Lengend Snippet: ICP0 induces degradation of PML-I regardless of mutated PML phosphorylation sites. HEp-2 were transfected with 900 ng of an empty vector (pGEM-3), a vector encoding ICP0, or a vector encoding the ICP0 mutant n212 and 100 ng of a plasmid encoding FLAG-, eCFP-tagged PML-I or a PML-I mutant. Twenty-four hours later, cells were lysed, resolved by SDS-PAGE, and analyzed by western blot with an anti-FLAG or anti-β-actin antibody.

Article Snippet: The transduced and infected cells were probed first with antibodies against ICP0 (H11060, Santa Cruz Biotechnology) and ICP4 (EastCoastBio) diluted in 5% rabbit serum in PBS and then probed with goat-anti-mouse IgG1 Dylight 594 and goat-anti-mouse IgG2b Dylight 488 as listed above for Sp100 and Daxx staining.

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Mutagenesis, SDS Page, Western Blot

Journal: Cells

Article Title: A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes

doi: 10.3390/cells3041131

Figure Lengend Snippet: Mutation of phosphorylation sites near the SIM compromises recruitment of PML-I to incoming HSV-1 genomes. HA-shPML cells transduced with FLAG-, eCFP-tagged PML-I or a PML-I mutant were infected with an ICP0-null virus at 0.1 PFU/cell. At 24 hpi, the cells were fixed and stained with antibody against ICP4 as a marker of viral DNA. Scale bar = 10 μm.

Article Snippet: The transduced and infected cells were probed first with antibodies against ICP0 (H11060, Santa Cruz Biotechnology) and ICP4 (EastCoastBio) diluted in 5% rabbit serum in PBS and then probed with goat-anti-mouse IgG1 Dylight 594 and goat-anti-mouse IgG2b Dylight 488 as listed above for Sp100 and Daxx staining.

Techniques: Mutagenesis, Phospho-proteomics, Transduction, Infection, Virus, Staining, Marker

Journal: Cells

Article Title: A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes

doi: 10.3390/cells3041131

Figure Lengend Snippet: Properties of PML-I and PML-I mutants.

Article Snippet: The transduced and infected cells were probed first with antibodies against ICP0 (H11060, Santa Cruz Biotechnology) and ICP4 (EastCoastBio) diluted in 5% rabbit serum in PBS and then probed with goat-anti-mouse IgG1 Dylight 594 and goat-anti-mouse IgG2b Dylight 488 as listed above for Sp100 and Daxx staining.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Different molecular forms of CYP2C8 in human liver samples. A, panels i–iii, representative immunoblots of mitochondrial and microsomal proteins (50 μg each) from human liver samples (Mt, mitochondrial fraction; Mc, microsomal fraction; HL, human liver used in labeling of some of the samples). Blots were developed with polyclonal antibodies to CYP2C8 (1:500 dilution, v/v) and TOM20 (1:2,000 dilution, v/v) and monoclonal antibody to cytochrome P450 reductase (CPR) (1:1,500 dilution, v/v). In addition to the full-length CYP2C8, a smaller form of 44 kDa (Var_3 (V3)) was seen predominantly in the mitochondrial fraction. The numbers in parentheses below the CYP2C8 immunoblot represent the ratios of Var_3 and full-length (Var_1) proteins in terms of band intensities. B, relative distribution of full-length CYP2C8 in mitochondria and microsomes of the liver samples analyzed in A. The percent distribution was calculated based on the densitometry of band intensities in A. Results represent averages from two blots.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Western Blot, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Identification of the 44-kDa species as a splice variant. A, protein sequence alignment of full-length (WT) CYP2C8 (marked 1) and its two splice variants (marked 2 and 3, respectively). Conserved residues in all three sequences are marked with asterisks. The reported Var_2 and Var_3 lack the N-terminal stretch of amino acids from the full-length protein. B, schematic representation of differential splicing of pre-mRNA for the generation of full-length CYP2C8 (Var_1) and Var_3 mRNAs. Ex, exon; aa, amino acids. C, DNA amplicons generated by RT-PCR of total RNAs using the common 3′- and 5′-primers were resolved on a 2% agarose gel (w/v) and stained with ethidium bromide. WT CYP2C8, Var_3 (V3), and a slow migrating minor component, Var_2 (V2), are shown for the liver samples analyzed in Fig. 1A. M, DNA marker. Relative band intensities of Var_3 and Var_1 amplicons are presented as ratios in parentheses below the gel patterns. C, panels i and ii, immunoblot analysis of mitochondrial proteins (50 μg each) from individual liver samples from Fig. 1A. Mitochondrial protein (Mito) from HepG2 cells expressing Var_3 cDNA was run alongside.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Variant Assay, Sequencing, Generated, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Marker, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: In silico analysis of WT CYP2C8 and CYP2C8 variant *3. A, three-dimensional structures of WT CYP2C8 (WT2C8; in yellow at left), Var_3 (V3-2C8; in pink at center), and Var_3 (V3) superimposed on WT (right). Heme is colored in red, and felodipine is colored blue. B, amino acid residues interacting with the heme (in red) through hydrogen-bonding interactions in WT CYP2C8 (at left) and Var_3 (at right). C, list of amino acid residues (one-letter notation) involved in anchoring the heme in the two molecular forms.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: In Silico, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Different subcellular targeting efficiencies of the three variant proteins. A, in vitro import of 35S-labeled translation products in isolated rat liver mitochondria. Panel i, 35S-labeled translation products of wild type (WT2C8), Var_2 (V2-2C8), and Var_3 (V3-2C8). Radiometric imaging of gels was performed to determine the level of import of input protein for each construct in trypsin-treated samples (T). The input protein level was considered to be 100% in each case. Panel ii, import of dihydrofolate reductase (DHFR) and SU9-dihydrofolate reductase (Su9-DHFR) proteins as negative and positive controls, respectively. The lanes marked “In” or “I” (for input) were loaded with 20% of the counts used for the import reactions. “C” represents control experiments in which total protein bound and imported into mitochondria is present, “T” represents trypsin-treated mitochondria in which only the protein imported into mitochondria is present. B, translocation of WT and variant 2 and 3 proteins in transiently transfected COS-7 cells. Panel i, total cell lysates (50 μg each) from transiently transfected COS-7 cells were resolved by 12% SDS (w/v)-PAGE and probed with antibodies to CYP2C8 and β-actin for assessing loading levels. Panel ii, mitochondria and microsomes were isolated from transfected COS-7 cells, and 50 μg of protein each was resolved by 12% SDS (w/v)-PAGE and probed with antibodies to CYP2C8, cytochrome P450 reductase (CPR), and cytochrome-c oxidase subunit I. Panel iii, relative resistance of mitochondrion-associated proteins to trypsin treatment (T). In some cases, mitochondria were lysed by treatment with 1% Triton X-100 (v/v) before trypsin treatment (TT). Proteins (50 μg each) were resolved by SDS-PAGE and probed with antibodies to CYP2C8 and TOM20 for immunoblot analysis. C, immunofluorescence microcopy of COS-7 cells transfected with WT (Var_1), Var_2, or Var_3 cDNA. Panel i, a–c, co-localization of CYP2C8 with a mitochondrial marker, cytochrome-c oxidase subunit I (CcOI). Cells were stained with a 1:1,000 dilution (v/v) of primary anti-goat antibody to CYP2C8 (green) (Abcam, Cambridge, MA) and co-stained with a 1:500 dilution (v/v) of cytochrome-c oxidase subunit I (red) (anti-mouse) antibody (Abcam). Panel ii, a–c, co-localization of CYP2C8 with microsomal membrane marker calreticulin. Cells were stained with CYP2C8 (green) as above and co-stained with a 1:500 dilution (v/v) of calreticulin (CRT) (anti-rabbit) antibody (red) (Santa Cruz Biotechnology, Santa Cruz, CA). The cells were subsequently incubated with secondary Alexa Fluor 546-conjugated anti-mouse and then anti-rabbit IgG and Alexa Fluor 488-conjugated anti-goat IgG and imaged through a confocal microscope. Numbers in the bottom panels indicate Pearson coefficients for coincidence calculated using Volocity 5.3 software.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Variant Assay, In Vitro, Labeling, Isolation, Imaging, Construct, Translocation Assay, Transfection, SDS Page, Western Blot, Immunofluorescence, Marker, Staining, Incubation, Microscopy, Software

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: The mitochondrial targeting efficiency of N-terminal signals of the full-length (Var_1; WT), Var_2 (V*2), and Var_3 (V*3) proteins The mitochondrial targeting efficiency of the three proteins was analyzed using the MitoProt II-v1.101 program. aa, amino acids.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Reconstitution of catalytic activity of purified WT CYP2C8. A, reconstitution of paclitaxel 6-hydroxylation activity was done with 0.2 nmol of purified CYP protein with or without 0.5 nmol of cytochrome P450 reductase (CPR), 0.4 nmol of purified Adx, 0.04 nmol of purified AdxR, and 10 μm paclitaxel in a 0.3-ml final volume as described under “Materials and Methods.” Montelukast (Mon) (5 μm) and inhibitory antibody to CYP2C8 (2C8Ab) (10 mg/ml) were used. Control ascites fluid (CAF; 10 mg/ml) was used as a negative control. B, reconstitution of dibenzylfluorescein oxidation was carried out essentially as described above in A. The activities in all cases represent the means ± S.E. (error bars) of three to five separate assays. Purified CYP2C8 was preincubated with inhibitors and control ascites fluid as described under “Materials and Methods.” ♦ in A indicates no detectable activity.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Activity Assay, Purification, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Subcellular distribution of CYP2C8 in stable HepG2 cells. A, immunoblot analysis of mitochondrial (Mt) and microsomal (Mc) fractions isolated from stable HepG2 cells with CYP2C8 antibody (middle panel). The blots were also probed with antibodies to TOM20 (bottom panel) as a mitochondrion-specific marker and NADPH-cytochrome P450 reductase (top panel) as a microsome-specific marker. Std, standard. B, membrane-extrinsic and -intrinsic nature of wild-type CYP2C8 (WT 2C8) and Var_3 (V3 2C8) in the mitochondrial (Mito) and microsomal (Micro) fractions was analyzed by the alkaline Na2CO3 extraction method described under “Materials and Methods.” The soluble (E) and insoluble (P) protein fractions were subjected to SDS-PAGE separation and probed with CYP2C8 antibody (2C8Ab) by immunoblot analysis. C, the relative levels of viral vector DNA (puromycin acetyltransferase gene) were determined by real time PCR using total cell DNA as template and the actin gene as an internal reference. D, spectrophotometric scans of CYP heme in the mitochondrial and microsomal fractions obtained from HepG2 stable cells expressing WT and Var_3 proteins. Fe2+-CO versus Fe2+ spectra were recorded as described under “Materials and Methods.” Abs, absorbance. E, relative CYP contents of mitochondria and microsomes from mock-, wild-type CYP2C8-, and CYP2C8 Var_3-expressing HepG2 cells. CYP content was measured by CO difference spectra as described under “Materials and Methods.” F, paclitaxel 6-hydroyxlation activity reconstituted with mitochondria and microsomes from stable HepG2 cells expressing WT CYP2C8 and Var_3 CYP2C8 and mock-transfected cells. Assays were carried out as described under “Materials and Methods.” G, ROS production in isolated mitochondria from stable HepG2 cell lines with or without treatment with the antioxidant N-acetylcysteine (NAC) or the inhibitors proadifen and montelukast. Mitochondria (50 μg each) were seeded in 96-well plates for ROS measurements using the 2′,7′-dichlorodihydrofluorescein (DCF) diacetate method as described under “Materials and Methods.” Results represent means ± S.E. (error bars) of three to four separate assays. * indicates a p value <0.05, and ** represents a p value <0.001. ♦ in F indicates no detectable activity. CPR, cytochrome P450 reductase.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Western Blot, Isolation, Marker, SDS Page, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Oxidation of arachidonic acid by mitochondria and microsomes isolated from stable HepG2 cells expressing Var_1 (WT) CYP2C8 and Var_3 proteins. A–D, the mitochondrial (Mt) and microsomal (MIC) proteins (300–500 μg) were assayed for arachidonic acid metabolism as described under “Materials and Methods” using 70 μm arachidonic acid as substrate. Inhibition studies were performed by preincubating enzymes with 5 μm montelukast at 37 °C for 20 min. Reactions were initiated by the addition of 1 mm NADPH and continued for 5 min at 37 °C in a shaking water bath, and the metabolites were extracted and analyzed as described under “Materials and Methods.” Four major arachidonate products (11,12-EET, 14,15-EET, 8,9-EET, and 20-HETE) were quantified as described under “Materials and Methods.” E, mitochondrial (Mito) proteins from Var_3 (V3)-expressing cells were reconstituted with or without added Adx/AdxR or added montelukast using arachidonic acid as substrate. In one case, purified Var_1 (V1) CYP2C8 was reconstituted with Adx/AdxR as described in Fig. 5. The total EET metabolites were quantified using the LC-MS method to ascertain the dependence of the enzyme on Adx/AdxR. The results represent means ± S.E. (error bars) of three independent assays. * indicates p < 0.05, and ** indicates p < 0.001. CPR, cytochrome P450 reductase.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Isolation, Expressing, Inhibition, Purification, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Respiratory dysfunction and ROS generation in cells treated with arachidonic acid. A, panels i–iii, the effect of arachidonic acid on respiration profile was measured using a Seahorse Bioscience XF24 extracellular flux analyzer. All parameters were analyzed using XF software and are displayed as oxygen consumption rates (pmol of O2/min/100 μg of protein) after normalizing for the protein concentration of each well. Panel i, basal OCR accounts for baseline rates of oxygen consumption. Panel ii, 2,4-dinitrophenol-mediated uncoupling generates maximal OCR. Panel iii, inhibition by oligomycin corresponds to ATP-linked OCR. Mean values ± S.E. (error bars) were calculated based on three separate measurements. B, effects of arachidonic acid (AA) on ROS production in stable HepG2 cells. Cells were grown with or without arachidonic acid for 24 h in 6-well plates, and the culture fluids were used for assaying the levels of H2O2 produced using the Amplex Red method as described under “Materials and Methods.” Results represent means ± S.E. (error bars) of three separate readings. * indicates a p value <0.05, and ** indicates a p value <0.001. ♦ in A (panel iii) indicates no detectable activity. V3, Var_3; 2C8, CYP2C8.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Software, Protein Concentration, Inhibition, Produced, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Different molecular forms of CYP2C8 in human liver samples. A, panels i–iii, representative immunoblots of mitochondrial and microsomal proteins (50 μg each) from human liver samples (Mt, mitochondrial fraction; Mc, microsomal fraction; HL, human liver used in labeling of some of the samples). Blots were developed with polyclonal antibodies to CYP2C8 (1:500 dilution, v/v) and TOM20 (1:2,000 dilution, v/v) and monoclonal antibody to cytochrome P450 reductase (CPR) (1:1,500 dilution, v/v). In addition to the full-length CYP2C8, a smaller form of 44 kDa (Var_3 (V3)) was seen predominantly in the mitochondrial fraction. The numbers in parentheses below the CYP2C8 immunoblot represent the ratios of Var_3 and full-length (Var_1) proteins in terms of band intensities. B, relative distribution of full-length CYP2C8 in mitochondria and microsomes of the liver samples analyzed in A. The percent distribution was calculated based on the densitometry of band intensities in A. Results represent averages from two blots.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Western Blot, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Identification of the 44-kDa species as a splice variant. A, protein sequence alignment of full-length (WT) CYP2C8 (marked 1) and its two splice variants (marked 2 and 3, respectively). Conserved residues in all three sequences are marked with asterisks. The reported Var_2 and Var_3 lack the N-terminal stretch of amino acids from the full-length protein. B, schematic representation of differential splicing of pre-mRNA for the generation of full-length CYP2C8 (Var_1) and Var_3 mRNAs. Ex, exon; aa, amino acids. C, DNA amplicons generated by RT-PCR of total RNAs using the common 3′- and 5′-primers were resolved on a 2% agarose gel (w/v) and stained with ethidium bromide. WT CYP2C8, Var_3 (V3), and a slow migrating minor component, Var_2 (V2), are shown for the liver samples analyzed in Fig. 1A. M, DNA marker. Relative band intensities of Var_3 and Var_1 amplicons are presented as ratios in parentheses below the gel patterns. C, panels i and ii, immunoblot analysis of mitochondrial proteins (50 μg each) from individual liver samples from Fig. 1A. Mitochondrial protein (Mito) from HepG2 cells expressing Var_3 cDNA was run alongside.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Variant Assay, Sequencing, Generated, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Marker, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: In silico analysis of WT CYP2C8 and CYP2C8 variant *3. A, three-dimensional structures of WT CYP2C8 (WT2C8; in yellow at left), Var_3 (V3-2C8; in pink at center), and Var_3 (V3) superimposed on WT (right). Heme is colored in red, and felodipine is colored blue. B, amino acid residues interacting with the heme (in red) through hydrogen-bonding interactions in WT CYP2C8 (at left) and Var_3 (at right). C, list of amino acid residues (one-letter notation) involved in anchoring the heme in the two molecular forms.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: In Silico, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Different subcellular targeting efficiencies of the three variant proteins. A, in vitro import of 35S-labeled translation products in isolated rat liver mitochondria. Panel i, 35S-labeled translation products of wild type (WT2C8), Var_2 (V2-2C8), and Var_3 (V3-2C8). Radiometric imaging of gels was performed to determine the level of import of input protein for each construct in trypsin-treated samples (T). The input protein level was considered to be 100% in each case. Panel ii, import of dihydrofolate reductase (DHFR) and SU9-dihydrofolate reductase (Su9-DHFR) proteins as negative and positive controls, respectively. The lanes marked “In” or “I” (for input) were loaded with 20% of the counts used for the import reactions. “C” represents control experiments in which total protein bound and imported into mitochondria is present, “T” represents trypsin-treated mitochondria in which only the protein imported into mitochondria is present. B, translocation of WT and variant 2 and 3 proteins in transiently transfected COS-7 cells. Panel i, total cell lysates (50 μg each) from transiently transfected COS-7 cells were resolved by 12% SDS (w/v)-PAGE and probed with antibodies to CYP2C8 and β-actin for assessing loading levels. Panel ii, mitochondria and microsomes were isolated from transfected COS-7 cells, and 50 μg of protein each was resolved by 12% SDS (w/v)-PAGE and probed with antibodies to CYP2C8, cytochrome P450 reductase (CPR), and cytochrome-c oxidase subunit I. Panel iii, relative resistance of mitochondrion-associated proteins to trypsin treatment (T). In some cases, mitochondria were lysed by treatment with 1% Triton X-100 (v/v) before trypsin treatment (TT). Proteins (50 μg each) were resolved by SDS-PAGE and probed with antibodies to CYP2C8 and TOM20 for immunoblot analysis. C, immunofluorescence microcopy of COS-7 cells transfected with WT (Var_1), Var_2, or Var_3 cDNA. Panel i, a–c, co-localization of CYP2C8 with a mitochondrial marker, cytochrome-c oxidase subunit I (CcOI). Cells were stained with a 1:1,000 dilution (v/v) of primary anti-goat antibody to CYP2C8 (green) (Abcam, Cambridge, MA) and co-stained with a 1:500 dilution (v/v) of cytochrome-c oxidase subunit I (red) (anti-mouse) antibody (Abcam). Panel ii, a–c, co-localization of CYP2C8 with microsomal membrane marker calreticulin. Cells were stained with CYP2C8 (green) as above and co-stained with a 1:500 dilution (v/v) of calreticulin (CRT) (anti-rabbit) antibody (red) (Santa Cruz Biotechnology, Santa Cruz, CA). The cells were subsequently incubated with secondary Alexa Fluor 546-conjugated anti-mouse and then anti-rabbit IgG and Alexa Fluor 488-conjugated anti-goat IgG and imaged through a confocal microscope. Numbers in the bottom panels indicate Pearson coefficients for coincidence calculated using Volocity 5.3 software.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Variant Assay, In Vitro, Labeling, Isolation, Imaging, Construct, Translocation Assay, Transfection, SDS Page, Western Blot, Immunofluorescence, Marker, Staining, Incubation, Microscopy, Software

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: The mitochondrial targeting efficiency of N-terminal signals of the full-length (Var_1; WT), Var_2 (V*2), and Var_3 (V*3) proteins The mitochondrial targeting efficiency of the three proteins was analyzed using the MitoProt II-v1.101 program. aa, amino acids.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Reconstitution of catalytic activity of purified WT CYP2C8. A, reconstitution of paclitaxel 6-hydroxylation activity was done with 0.2 nmol of purified CYP protein with or without 0.5 nmol of cytochrome P450 reductase (CPR), 0.4 nmol of purified Adx, 0.04 nmol of purified AdxR, and 10 μm paclitaxel in a 0.3-ml final volume as described under “Materials and Methods.” Montelukast (Mon) (5 μm) and inhibitory antibody to CYP2C8 (2C8Ab) (10 mg/ml) were used. Control ascites fluid (CAF; 10 mg/ml) was used as a negative control. B, reconstitution of dibenzylfluorescein oxidation was carried out essentially as described above in A. The activities in all cases represent the means ± S.E. (error bars) of three to five separate assays. Purified CYP2C8 was preincubated with inhibitors and control ascites fluid as described under “Materials and Methods.” ♦ in A indicates no detectable activity.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Activity Assay, Purification, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Subcellular distribution of CYP2C8 in stable HepG2 cells. A, immunoblot analysis of mitochondrial (Mt) and microsomal (Mc) fractions isolated from stable HepG2 cells with CYP2C8 antibody (middle panel). The blots were also probed with antibodies to TOM20 (bottom panel) as a mitochondrion-specific marker and NADPH-cytochrome P450 reductase (top panel) as a microsome-specific marker. Std, standard. B, membrane-extrinsic and -intrinsic nature of wild-type CYP2C8 (WT 2C8) and Var_3 (V3 2C8) in the mitochondrial (Mito) and microsomal (Micro) fractions was analyzed by the alkaline Na2CO3 extraction method described under “Materials and Methods.” The soluble (E) and insoluble (P) protein fractions were subjected to SDS-PAGE separation and probed with CYP2C8 antibody (2C8Ab) by immunoblot analysis. C, the relative levels of viral vector DNA (puromycin acetyltransferase gene) were determined by real time PCR using total cell DNA as template and the actin gene as an internal reference. D, spectrophotometric scans of CYP heme in the mitochondrial and microsomal fractions obtained from HepG2 stable cells expressing WT and Var_3 proteins. Fe2+-CO versus Fe2+ spectra were recorded as described under “Materials and Methods.” Abs, absorbance. E, relative CYP contents of mitochondria and microsomes from mock-, wild-type CYP2C8-, and CYP2C8 Var_3-expressing HepG2 cells. CYP content was measured by CO difference spectra as described under “Materials and Methods.” F, paclitaxel 6-hydroyxlation activity reconstituted with mitochondria and microsomes from stable HepG2 cells expressing WT CYP2C8 and Var_3 CYP2C8 and mock-transfected cells. Assays were carried out as described under “Materials and Methods.” G, ROS production in isolated mitochondria from stable HepG2 cell lines with or without treatment with the antioxidant N-acetylcysteine (NAC) or the inhibitors proadifen and montelukast. Mitochondria (50 μg each) were seeded in 96-well plates for ROS measurements using the 2′,7′-dichlorodihydrofluorescein (DCF) diacetate method as described under “Materials and Methods.” Results represent means ± S.E. (error bars) of three to four separate assays. * indicates a p value <0.05, and ** represents a p value <0.001. ♦ in F indicates no detectable activity. CPR, cytochrome P450 reductase.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Western Blot, Isolation, Marker, SDS Page, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Oxidation of arachidonic acid by mitochondria and microsomes isolated from stable HepG2 cells expressing Var_1 (WT) CYP2C8 and Var_3 proteins. A–D, the mitochondrial (Mt) and microsomal (MIC) proteins (300–500 μg) were assayed for arachidonic acid metabolism as described under “Materials and Methods” using 70 μm arachidonic acid as substrate. Inhibition studies were performed by preincubating enzymes with 5 μm montelukast at 37 °C for 20 min. Reactions were initiated by the addition of 1 mm NADPH and continued for 5 min at 37 °C in a shaking water bath, and the metabolites were extracted and analyzed as described under “Materials and Methods.” Four major arachidonate products (11,12-EET, 14,15-EET, 8,9-EET, and 20-HETE) were quantified as described under “Materials and Methods.” E, mitochondrial (Mito) proteins from Var_3 (V3)-expressing cells were reconstituted with or without added Adx/AdxR or added montelukast using arachidonic acid as substrate. In one case, purified Var_1 (V1) CYP2C8 was reconstituted with Adx/AdxR as described in Fig. 5. The total EET metabolites were quantified using the LC-MS method to ascertain the dependence of the enzyme on Adx/AdxR. The results represent means ± S.E. (error bars) of three independent assays. * indicates p < 0.05, and ** indicates p < 0.001. CPR, cytochrome P450 reductase.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Isolation, Expressing, Inhibition, Purification, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Respiratory dysfunction and ROS generation in cells treated with arachidonic acid. A, panels i–iii, the effect of arachidonic acid on respiration profile was measured using a Seahorse Bioscience XF24 extracellular flux analyzer. All parameters were analyzed using XF software and are displayed as oxygen consumption rates (pmol of O2/min/100 μg of protein) after normalizing for the protein concentration of each well. Panel i, basal OCR accounts for baseline rates of oxygen consumption. Panel ii, 2,4-dinitrophenol-mediated uncoupling generates maximal OCR. Panel iii, inhibition by oligomycin corresponds to ATP-linked OCR. Mean values ± S.E. (error bars) were calculated based on three separate measurements. B, effects of arachidonic acid (AA) on ROS production in stable HepG2 cells. Cells were grown with or without arachidonic acid for 24 h in 6-well plates, and the culture fluids were used for assaying the levels of H2O2 produced using the Amplex Red method as described under “Materials and Methods.” Results represent means ± S.E. (error bars) of three separate readings. * indicates a p value <0.05, and ** indicates a p value <0.001. ♦ in A (panel iii) indicates no detectable activity. V3, Var_3; 2C8, CYP2C8.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Software, Protein Concentration, Inhibition, Produced, Activity Assay

Journal: International Journal of Cell Biology

Article Title: Antiproliferative Factor-Induced Changes in Phosphorylation and Palmitoylation of Cytoskeleton-Associated Protein-4 Regulate Its Nuclear Translocation and DNA Binding

doi: 10.1155/2012/150918

Figure Lengend Snippet: Surface-labeled CKAP4 translocates from the plasma membrane into the nucleus following APF exposure . (a) HeLa cell-surface proteins were labeled with Sulfo NHS-biotin as described in . Following exposure to 20 nM APF for 24 hours (or no treatment), the cells were harvested and the nuclear protein fraction was isolated (Pierce NE-PER), separated by SDS-PAGE, and transferred to nitrocellulose. The membrane was then probed with streptavidin-HRP (1 : 5000; Pierce) to bind biotinylated proteins, and the signal was detected by ECL (Pierce). Following detection of the biotinylated proteins from the nucleus, the (streptavidin) HRP on the membrane was inactivated by incubating the blot in PBS containing 3% H 2 O 2 and 1% sodium azide. The same membrane was then reprobed with antibodies to CKAP4 (“anti-CLIMP-63”, diluted 1 : 1000, Alexis Biochemicals) and fibrillarin (a nuclear marker and loading control; Abcam; diluted 1 : 1000). (b) HeLa cells were treated with APF (20 nM) for 24 hours, which resulted in a significant increase in the abundance of CKAP4 in the nucleus compared to control samples. Treated cells were harvested and the nuclear and cytosolic fractions were isolated and separated by SDS-PAGE as described in . Protein expression was analyzed by Western Blotting with antibodies for β -tubulin (diluted 1 : 1000, Abcam; loading control for the nonnuclear fraction), CKAP4 (“anti-CLIMP-63”, diluted 1 : 1000, Alexis Biochemicals), and fibrillarin (diluted 1 : 1000, Abcam; loading control and specific marker for the nuclear fraction), and then with an HRP-conjugated anti-mouse secondary antibody (1 : 20000; ThermoFisher Scientific). The proteins were detected by ECL (Pierce) with multiple exposures to film. The integrated density of the bands on the film was measured using ImageJ. Exposure times were controlled to ensure that the signals on film were not saturated. (c) The nuclear/cytosolic ratio represents the relative distribution of CKAP4 in the nuclear versus cytosolic fractions extracted from cells treated with or without APF. CKAP4 abundance in the APF-treated and control samples were normalized for loading to β -tubulin for the nonnuclear fractions and to fibrillarin for the nuclear fractions. The nuclear/cytosolic ratio for CKAP4 in the APF and control samples was determined from these normalized values. The standard deviation describes the variability among the normalized, nuclear, and cytosolic ratios from three independent experiments. A two tailed, paired t -test of the two data arrays (plus APF and control) indicate that the difference between these ratios is significant ( P = 0.01; n = 3). Cells treated with APF stop dividing, so the 10 cm dishes containing control and APF treated cells contained fewer cells (and protein) at the end of the experiment, normalizing the CKAP4 signals to loading controls corrected for this disparity. Fibrillarin is a well-characterized nuclear marker that is also known to localize to nucleoli. The data shown are representative of four independent experiments.

Article Snippet: HeLa cells were serum-starved overnight and then treated with 20 nM synthetic APF (Peptides International) for 24 hours.

Techniques: Labeling, Isolation, SDS Page, Marker, Expressing, Western Blot, Standard Deviation, Two Tailed Test

Journal: International Journal of Cell Biology

Article Title: Antiproliferative Factor-Induced Changes in Phosphorylation and Palmitoylation of Cytoskeleton-Associated Protein-4 Regulate Its Nuclear Translocation and DNA Binding

doi: 10.1155/2012/150918

Figure Lengend Snippet: CKAP4 phosphorylation and palmitoylation regulate CKAP4 trafficking . CKAP4 mutants that mimic constitutive depalmitoylation and various states of serine [ , , ] phosphorylation were generated to determine the effect of these two posttranslational modifications on the subcellular distribution of CKAP4 in response to APF (see ). HeLa cells were transfected for 24–36 hours with the construct indicated to the left of each panel. Cells were serum starved for 6 hours and then treated with APF (20 nM) for 18–24 hours. Subsequently, the cells were fixed in 4% buffered paraformaldehyde, permeabilized with 0.1% Triton X-100, and immunostained for (1) β -tubulin (red; TRITC) and (2) V5 (green: FITC) (as described in ) to distinguish the transfected, V5-epitope tagged WT (a) and mutant versions of CKAP4 from endogenous CKAP4 (b)–(d). Mutant versions of CKAP4 that cannot be palmitoylated (C100S) or phosphorylated (SΔA) do not translocate to the nucleus in response to APF. (e) Those that mimic phosphorylation (SΔE) translocate to the nucleus in response to APF. (f) CKAP4C100SSΔE, which is constitutively depalmitoylated and phosphomimicking, is expressed primarily in the nucleus. Images taken in each channel were superimposed to illustrate the distribution of mutant CKAP4 with respect to the cytoskeleton. The cells were imaged by epifluorescence or confocal microscopy at 60X and 63X, respectively, (Scale bars = 25 microns).

Article Snippet: HeLa cells were serum-starved overnight and then treated with 20 nM synthetic APF (Peptides International) for 24 hours.

Techniques: Generated, Transfection, Construct, Mutagenesis, Confocal Microscopy

Journal: International Journal of Cell Biology

Article Title: Antiproliferative Factor-Induced Changes in Phosphorylation and Palmitoylation of Cytoskeleton-Associated Protein-4 Regulate Its Nuclear Translocation and DNA Binding

doi: 10.1155/2012/150918

Figure Lengend Snippet: APF induces serine phosphorylation of CKAP4 . (a) APF treatment induces a significant increase in serine phosphorylation of CKAP4 as demonstrated by immunoprecipitation of CKAP4 followed immunoblotting to detect phosphoserine. Whole cell lysates (500 μ g) from HeLa cells treated with 20 nM APF or serum-starved controls were immunoprecipitated with CKAP4 antibody (Alexis) overnight at 4°C. Samples were then bound to Protein A, washed (4X with RIPA/Empigen buffer), eluted (4X LDS sample buffer; Invitrogen), boiled at 95°C for 5 min and resolved on a 4–12% Bis-Tris gel and transferred to a nitrocellulose membrane under reducing conditions. Western Blot analysis for pSer detected phospho-serine using primary (Invitrogen and secondary antibodies (goat anti-rabbit HRP-labeled antibody; Pierce)) developed with Enhanced Chemiluminescence reagent (Pierce) and exposed to film. The membrane was stripped with Restore Stripping Buffer (Pierce) and reprobed for CKAP4 (Alexis) to normalize the phosphoserine signal to the amount of immunoprecipitated CKAP4. (b) Densitometric analysis of the immunoreactive bands was done using ImageJ, and the ratios of phosphorylated to nonphosphorylated CKAP4 were determined.

Article Snippet: HeLa cells were serum-starved overnight and then treated with 20 nM synthetic APF (Peptides International) for 24 hours.

Techniques: Immunoprecipitation, Western Blot, Labeling, Stripping Membranes

Journal: International Journal of Cell Biology

Article Title: Antiproliferative Factor-Induced Changes in Phosphorylation and Palmitoylation of Cytoskeleton-Associated Protein-4 Regulate Its Nuclear Translocation and DNA Binding

doi: 10.1155/2012/150918

Figure Lengend Snippet: Nuclear CKAP4 is phosphorylated following APF-induced translocation . (a) CKAP4 in the nuclear fraction was phosphorylated to the same degree in APF-treated and control cells as demonstrated by metabolic labeling with γ 32 P-ATP. The ratio of the CKAP4 phosphorylation signal over the corresponding CKAP4 Western Blot signal was approximately the same for both (1.00 versus 0.975; APF treated versus control). HeLa cells at ~80% confluence in 10 cm dishes were serum starved for 3 hours then 150 μ Ci γ 32 P-ATP was added to each dish for 1 additional hour. Then the cells were either exposed to APF (20 nM; 24 hours) or left in serum-free medium (control) for 24 hours. At the end of 24 hours, the cells were harvested, washed three times in ice-cold PBS, and the nuclear and cytoplasmic fractions were isolated using the NE-PER (Pierce). Equal quantities of each fraction were separated by SDS-PAGE and transferred to nitrocellulose. The membrane was incubated overnight at 4°C in α -CKAP4 antibody (1 : 500) in TBST and 1% milk, washed and incubated for six hours at 4°C in a-mouse, HRP-conjugated secondary antibody (Pierce; 1 : 20,000) in TBST and 1% milk. CKAP4 bands were detected by enhanced chemiluminescence (ECL; Pierce; 20 second exposure; left panel). Following the Western Blot, the membrane was rinsed in 1% H 2 O 2 for 1 minute to eliminate the chemiluminescent signal then wrapped in plastic wrap and exposed to film for 12 hours to detect the phosphorylated proteins. Densitometric analysis of the immunoreactive bands was done using ImageJ, and the ratios of phosphorylated to nonphosphorylated CKAP4 were determined. APF increases the association of CKAP4 with nucleoli. (b) and (c) Western Blot analysis shows that treatment of HeLa cells with APF (20 nM) increased the association of endogenous CKAP4 with the nucleolar fraction, and that the constitutively depalmitoylated and phosphomimicking CKAP4 mutant, CKAP4C100S/SΔE, associated with the nucleolar fraction to a greater extent than endogenous CKAP4 isolated from APF-treated cells. Nucleoli were isolated using a variation on the method published by Busch and coworkers as described by the Angus Lamond lab (University of Dundee, UK). The nucleolar proteins were separated by SDS-PAGE and Western Blotted for CKAP4 ( α -V5 in the case of CKAP4C100S/SΔE) and fibrillarin. The bands were detected on film by ECL and quantified using ImageJ. The CKAP4 signal in each lane of the Western Blot was normalized to the fibrillarin band in the same lane. The normalized value for CKAP4 from control cells was set to 1 and the other values were set relative to control. The values in the graph are means and SD. “*” indicates that the means of the values were significantly different than serum-starved control when evaluated using the students t -test (two-tailed. Serum starved versus APF treated P = 0.018; serum starved versus CKAP4C100S/SΔE P = 0.002; CKAP4C100S/SΔE versus APF treated, P = 0.008; n = 3 for each).

Article Snippet: HeLa cells were serum-starved overnight and then treated with 20 nM synthetic APF (Peptides International) for 24 hours.

Techniques: Translocation Assay, Labeling, Western Blot, Isolation, SDS Page, Incubation, Mutagenesis, Two Tailed Test