Journal: FEBS letters

Article Title: Myosin light chains are not a physiological substrate of AMPK in the control of cell structure changes.

doi: 10.1016/j.febslet.2008.11.022

Figure Lengend Snippet: Fig. 1. Phosphorylation of (A) MLC peptide and (B) SAMS peptide by the AMPK and smMLCK preparations in vitro. Initial rates of 32P-incorporation by 0.07 lg of rat liver AMPK (4), 0.07 lg of recombinant AMPK (h) or 1 lg of smMLCK (s) into the MLC peptide were 137 ± 18, 105 ± 7 and 194 ± 12 lUnits/50 ll of reaction mixture, respectively, and into the SAMS peptide were 215 ± 9, 201 ± 26 and 2.3 ± 1.1 lUnits/50 ll of reaction mixture, respectively.

Article Snippet: CaM, antifull-length smMLCK antibody (clone K36, Sigma), GST-human acetyl-CoA carboxylase (ACC)2, purified rat liver AMPK (Upstate), anti-rabbit Immunoglobulin G conjugated to IR dye 800 (Rockland Inc., Philadelphia, PA), anti-sheep Immunoglobulin G conjugated to Alexa 680 (Molecular Probes), anti-phospho-Thr172 AMPK a-subunit and anti-phospho Ser19 MLC antibodies (Bioké), anti-phospho Ser221 ACC2, anti-full-length ACC2 antibodies (Upstate), anti-fulllength MLC antibody (Sigma) and enhanced chemiluminescence reagents (Thermofisher) were from the indicated sources.

Techniques: Phospho-proteomics, In Vitro, Recombinant

Journal: FEBS letters

Article Title: Myosin light chains are not a physiological substrate of AMPK in the control of cell structure changes.

doi: 10.1016/j.febslet.2008.11.022

Figure Lengend Snippet: Fig. 2. Phosphorylation of (A) GST-ACC2 peptide and (B) MLC by the AMPK and smMLCK preparations in vitro. Initial rates of 32P-incorporation by 0.07 lg of rat liver AMPK (4), 0.07 lg of recombinant AMPK (h) or 1 lg of smMLCK (s) into GST- ACC2 were 10.5 ± 1.3, 5.7 ± 0.9 and 0.3 ± 0.1 lUnits/50 lL of reaction mixture, respectively, and into MLC were 12.0 ± 0.9, 1.3 ± 0.4 and >109 ± 4 lUnits/50 ll of reaction mixture, respectively.

Article Snippet: CaM, antifull-length smMLCK antibody (clone K36, Sigma), GST-human acetyl-CoA carboxylase (ACC)2, purified rat liver AMPK (Upstate), anti-rabbit Immunoglobulin G conjugated to IR dye 800 (Rockland Inc., Philadelphia, PA), anti-sheep Immunoglobulin G conjugated to Alexa 680 (Molecular Probes), anti-phospho-Thr172 AMPK a-subunit and anti-phospho Ser19 MLC antibodies (Bioké), anti-phospho Ser221 ACC2, anti-full-length ACC2 antibodies (Upstate), anti-fulllength MLC antibody (Sigma) and enhanced chemiluminescence reagents (Thermofisher) were from the indicated sources.

Techniques: Phospho-proteomics, In Vitro, Recombinant

Journal: FEBS letters

Article Title: Myosin light chains are not a physiological substrate of AMPK in the control of cell structure changes.

doi: 10.1016/j.febslet.2008.11.022

Figure Lengend Snippet: Fig. 3. Effect of H1152 on A769662-induced AMPK Thr172, ACC Ser221 and MLC Ser19 phosphorylation in MDCK cells. MDCK cells were incubated in the presence or absence of 20 lM A769662 for 30 min with or without 60 min of pre-treatment with 1 lM H1152 Rho kinase inhibitor. Cell lysates were analyzed by immunoblotting with anti- phospho Thr172 AMPK a-subunit, anti-phospho Ser221 ACC or anti-phospho Ser19 MLC antibodies along with the appropriate anti-full length protein antibodies as loading controls for detection by chemiluminescence and scanning densitometry. *Indicates a significant difference (P < 0.05, unpaired t-test) with respect to the value corresponding to treatment without inhibitor.

Article Snippet: CaM, antifull-length smMLCK antibody (clone K36, Sigma), GST-human acetyl-CoA carboxylase (ACC)2, purified rat liver AMPK (Upstate), anti-rabbit Immunoglobulin G conjugated to IR dye 800 (Rockland Inc., Philadelphia, PA), anti-sheep Immunoglobulin G conjugated to Alexa 680 (Molecular Probes), anti-phospho-Thr172 AMPK a-subunit and anti-phospho Ser19 MLC antibodies (Bioké), anti-phospho Ser221 ACC2, anti-full-length ACC2 antibodies (Upstate), anti-fulllength MLC antibody (Sigma) and enhanced chemiluminescence reagents (Thermofisher) were from the indicated sources.

Techniques: Phospho-proteomics, Incubation, Western Blot

Journal: bioRxiv

Article Title: DNA damage is a major cause of sequencing errors, directly confounding variant identification

doi: 10.1101/070334

Figure Lengend Snippet: A. Principle of the GIV score : Illumina adaptors are directional in nature, enabling consistent paired end sequencing within clusters. This property results in sequencing of the original strand orientation in the R1 reads (from the P5 adaptor) whereas the reverse complement orientation is read in the R2 reads (from the P7 adaptor). As damage affects only one base of a pair, damage such as 8-oxo-dG leads to an excess of G to T transversion errors when R1 is mapped to a reference genome, whereas, the R2 reads will show an excess of the reverse complement of G to T, i.e. C to A transversion errors, instead. As a consequence, there is a global imbalance in the number of G to T variants in R1 compared to R2 sequences. This imbalance is specific to damage and is the basis of the GIV score (see ). B. Overall fraction of G to T variants (normalized to the total number of G) for R1 and the reverse complement of R2 sequences. Different buffers were used during acoustic shearing (x-axis). Data in red were from samples that were not repaired and data in blue were from samples that were repaired. Each point corresponds to a random sampling of 2 million sequence positions. All samples are derived from the same human genomic DNA. C. Variant profile: The fraction of G to T and C to A variants in R1 and R2 sequences are plotted as a function of the read (R1 or R2) and the positions on the read (in bp). D. Correlation (R=0.97) between the degree of damage that is repaired by the DNA repair enzyme cocktail and GIV for G to T variant (GIV G_T ) and T to A variant (GIV T_A ).

Article Snippet: To introduce oxidative damage, purified human liver genomic DNA samples (BioChain Institute, Inc.) were fragmented by shearing in 1x TE (10 mM Tris pH 8, 1 mM EDTA), 0.1x TE (1 mM Tris pH 8, 0.1 mM EDTA) or water using a Covaris S2 to produce 200 bp average fragment size as determined by Bioanalyzer (Agilent Technologies) analysis.

Techniques: Sequencing, Sampling, Derivative Assay, Variant Assay

Journal: bioRxiv

Article Title: DNA damage is a major cause of sequencing errors, directly confounding variant identification

doi: 10.1101/070334

Figure Lengend Snippet: Genomic DNA was sheared in 0.1xTE buffer and subjected to library preparation and cancer panel target enrichment. DNA repair treatment was performed postshearing and prior to library preparation. The control represents untreated DNA. A. Overall G to T variant profiles across reads R1 and R2 with (red) and without (blue) repair treatment. B. Variant spectrum at a frequency less than 1% (left panel), 1 to 5% (center left panel), 6 to 10% (center right panel) and more than 10% (right panel). C. Same as for B except that only R1 reads were used for variant calling.

Article Snippet: To introduce oxidative damage, purified human liver genomic DNA samples (BioChain Institute, Inc.) were fragmented by shearing in 1x TE (10 mM Tris pH 8, 1 mM EDTA), 0.1x TE (1 mM Tris pH 8, 0.1 mM EDTA) or water using a Covaris S2 to produce 200 bp average fragment size as determined by Bioanalyzer (Agilent Technologies) analysis.

Techniques: Variant Assay

Journal: The Journal of biological chemistry

Article Title: Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter.

doi: 10.1074/jbc.272.51.32500

Figure Lengend Snippet: FIG. 1. Analysis of rat AR promoter constructs in transfected rat liver cells. A, scheme of rat AR promoter con- structs used in the transfection experi- ments. B, the luciferase activity (relative luciferase activity) from rat liver cells transfected with constructs 1 to 9 and subsequently grown in isotonic medium (nonstress, open bar) or hypertonic me- dium (stress, filled bar) for 18 h. Error bars indicate standard deviation (S.D.). C, luciferase activity 6 S.D. under isotonic or hypertonic conditions. pX/p1 indicates the increase over p1 luciferase activity. Ratio H/I indicates the ratio between hy- pertonic and isotonic activity; n indicates the number of replicates.

Article Snippet: Cell Culture, Transfection, and Osmotic Shock—A normal rat liver cell line (Clone 9, ATCC, Rockville, MD) which expresses AR and responds to osmotic stress (data not shown) was cultured in 35-mm * The costs of publication of this article were defrayed in part by the payment of page charges.

Techniques: Construct, Transfection, Luciferase, Activity Assay, Standard Deviation

Journal: The Journal of biological chemistry

Article Title: Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter.

doi: 10.1074/jbc.272.51.32500

Figure Lengend Snippet: FIG. 2. Exonuclease III-mediated DNA footprinting of promoter region necessary for osmotic response. Nuclei isolated from rat liver cells transfected with luciferase reporter construct 4 were treated with ExoIII as described under “Experimental Procedures.” Purified DNA was then subjected to linear PCR. Lane 1, linear PCR with primer 1 on HindIII-digested control template; 2, linear PCR with primer 1 on HindIII and ExoIII-digested transient template; 3, linear PCR with primer 2 on Asp718-digested control template; 4, linear PCR with primer 2 on Asp718 and ExoIII-digested transient template. The two horizontal complementary nucleotide sequences contain the primer locations and the ExoIII protected sequence is indicated by the dotted vertical lines. Sequencing reactions (A, C, G, T) were performed with the same primers used for linear PCR.

Article Snippet: Cell Culture, Transfection, and Osmotic Shock—A normal rat liver cell line (Clone 9, ATCC, Rockville, MD) which expresses AR and responds to osmotic stress (data not shown) was cultured in 35-mm * The costs of publication of this article were defrayed in part by the payment of page charges.

Techniques: DNA Footprinting, Isolation, Transfection, Luciferase, Construct, Purification, Control, Sequencing

Journal: The Journal of biological chemistry

Article Title: Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter.

doi: 10.1074/jbc.272.51.32500

Figure Lengend Snippet: FIG. 3. Dimethyl sulfate-mediated in vivo DNA footprinting of the promoter region involved in increased constitutive activity and osmotic response. Dimethyl sulfate-mediated in vivo DNA footprinting was performed on rat liver cells cultured under normal conditions or under osmotic stress as described under “Experimental Procedures.” Lane 1, LM-PCR of DNA from cells in hyperosmotic medium for 3 h (stress 3 h/in vivo); 2, LM-PCR of DNA from cells in hyperosmotic medium for 30 min (stress 309/in vivo); 3, LM-PCR of DNA from cells in normal osmotic medium (nonstress/in vivo); 4, LM-PCR of in vitro methylated DNA (G). Horizontal nucleotide sequence shows the primer 2 location; dotted line indicates the ExoIII stop location and short solid lines indicate guanosine band pattern. Arrows A and B indicate the positions of methylation- protected guanosines bands. Bold underline G indicates the same protected guanosines in the promoter sequence.

Article Snippet: Cell Culture, Transfection, and Osmotic Shock—A normal rat liver cell line (Clone 9, ATCC, Rockville, MD) which expresses AR and responds to osmotic stress (data not shown) was cultured in 35-mm * The costs of publication of this article were defrayed in part by the payment of page charges.

Techniques: In Vivo, DNA Footprinting, Activity Assay, Cell Culture, In Vitro, Methylation, Sequencing

Journal: The Journal of biological chemistry

Article Title: Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter.

doi: 10.1074/jbc.272.51.32500

Figure Lengend Snippet: FIG. 4. EMSA of the 32-bp exonuclease III-protected region with whole cell extract from rat liver cells. The ExoIII-protected 32-bp region was used as a probe to confirm the factor binding and to determine the effect of protein binding by mutation. Lane 1, probe A (no extract added); lane 2, probe A/normal osmolarity (A/n); lane 3, probe A/hyper-osmolarity for 30 min (A/s(309)); lane 4, probe A/hyper-osmolarity for 3 h (A/s(3 h)); lane 5, probe A/normal osmolarity/100 3 cold probe A (A/n comp:A); lane 6, probe A/hyper-osmolarity for 30 min/100 3 cold probe A (A/s(309) comp:A); lane 7, probe A/hyper-osmolarity for 3 h/100 3 cold probe A (A/s(3 h) comp:A); lane 8, probe A8/normal osmolarity (A8/n); lane 9, probe A8 hyper-osmolarity for 30 min (A8/s(309); lane 10, probe A8 hyper-osmolarity for 3 h (A8/s(3 h)); lane 11, probe A/normal osmolarity/100 3 cold probe A8 (A/n comp:A8); lane 12, probe A/hyper-osmolarity for 30 min/100 3 cold probe A8 (A/s(309) Comp:A8); lane 13, probe A/hyper- osmolarity for 3 h/100 3 cold probe A8 (A/s(3 h) Comp:A8). Arrows A-F indicate the complexes. Probe A contains the ExoIII-protected 32-bp region, Probe B is the same as Probe A except for 8 nucleotide replacements. Protected guanosines by dimethyl sulfate methylation (Fig. 3) in Probe A sequence are indicated by an asterisk and the mutations made for probe A8 are indicated by underline. Samples in lanes 1–7 and 11–13 were incubated with probe A.

Article Snippet: Cell Culture, Transfection, and Osmotic Shock—A normal rat liver cell line (Clone 9, ATCC, Rockville, MD) which expresses AR and responds to osmotic stress (data not shown) was cultured in 35-mm * The costs of publication of this article were defrayed in part by the payment of page charges.

Techniques: Binding Assay, Protein Binding, Mutagenesis, Methylation, Sequencing, Incubation