Journal: The Journal of biological chemistry

Article Title: Hepatitis C virus core protein binds to a DEAD box RNA helicase.

doi: 10.1074/jbc.274.22.15751

Figure Lengend Snippet: FIG. 1. Primary structures of DBX, PL10, and Ded1 and their interac- tions with HCV core protein in the yeast two-hybrid assay. A, alignment of deduced amino acid sequences of DBX (Gen- Banky accession number AF000982), PL10 (GenBanky accession number J04847), and Ded1p (GenBanky accession number X57278) is shown. Identical amino acids are shown as white on cyan. Conserved substi- tutions are shown as black on magenta. Dots represent gaps to optimize alignments, which were obtained using the Pileup pro- gram. B, two-hybrid assays showing interac- tion of HCV core protein with DBX and PL10 but not with Ded1p. Yeast strain Y190 was co-transformed with a plasmid express- ing the cytoplasmic domain of HCV fused to the GAL4 DNA binding domain and plas- mids expressing either a portion of DBX or the corresponding portions of PL10 or Ded1p fused to the GAL4 transcriptional activation domain. Transformants giving b-galactosid- ase activity (positive interactions) are blue. Control reactions of DBX, PL10, and Dep1p GAL 4 activation domain fusion proteins with GAL4 DNA binding domain alone were negative (data not shown).

Article Snippet: The amplified DNA was cloned into the GAL4 DNA binding domain fusion vector pAS2–1 (CLONTECH) to yield pAS2–1-HCV-core1–123.

Techniques: Y2H Assay, Transformation Assay, Plasmid Preparation, Binding Assay, Expressing, Activation Assay, Activity Assay, Control

Journal:

Article Title: Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase ?

doi:

Figure Lengend Snippet: Oligonucleotide primers and deletion strategy. (A) Oligonucleotides used for the construction of plasmid pAS2-p79, plasmids with p79 deletions (pAS2-p79Δ0, pAS2-p79Δ1, pAS2-p79Δ2, pAS2-p79Δ3, pAS2-p79Δ4, pAS2-p79Δ5 and pAS2-p79Δ6) and plasmids containing p167 deletions (pACT-p167δ3 and pACT-p167δ4). (B) Schematic representation of p79 deletion constructs. The deletion fragments were generated by PCR amplifications as described in Materials and Methods.

Article Snippet: The PCR-amplified DNA was cloned into pAS2-1 vector (Clontech Laboratories) at Nco I and Sal I sites, and the resultant plasmid pAS2p79 was transformed in the budding yeast strain, Y190 (in GAL4 2H-2; MAT a , ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4 Δ, gal80 Δ, cyhr2, LYS2::GAL1 UAS - HIS3 TATA - HIS3, MEL1 URA3:: GAL1UAS-GAL1 TATA - lacZ ).

Techniques: Plasmid Preparation, Construct, Generated

Journal:

Article Title: Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase ?

doi:

Figure Lengend Snippet: Interactions between p167 and p79 subunits. (A) The Y190 yeast strain was co-transformed with p167 deletions δ1/δ2 with pAS2-p79, where δ1 and δ2 refer to plasmids pACT-p167δ1 and pACT-p167δ2, respectively. The colonies were picked and re-streaked on SD- SD-Trp–/Leu– (a) and SD-Trp–/Leu–/His–+3AT plates (b) and incubated for 3 and 4 days, respectively. (a′) and (b′) represent the results from the X-gal colony filter-lift assay for the respective plates. (B) β-Galactosidase activity was determined quantitatively using the liquid culture assay as described in Materials and Methods for the interaction between pACT-p167δ1/δ2 and pAS2-p79.

Article Snippet: The PCR-amplified DNA was cloned into pAS2-1 vector (Clontech Laboratories) at Nco I and Sal I sites, and the resultant plasmid pAS2p79 was transformed in the budding yeast strain, Y190 (in GAL4 2H-2; MAT a , ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4 Δ, gal80 Δ, cyhr2, LYS2::GAL1 UAS - HIS3 TATA - HIS3, MEL1 URA3:: GAL1UAS-GAL1 TATA - lacZ ).

Techniques: Transformation Assay, Incubation, Activity Assay

Journal:

Article Title: Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase ?

doi:

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: The PCR-amplified DNA was cloned into pAS2-1 vector (Clontech Laboratories) at Nco I and Sal I sites, and the resultant plasmid pAS2p79 was transformed in the budding yeast strain, Y190 (in GAL4 2H-2; MAT a , ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4 Δ, gal80 Δ, cyhr2, LYS2::GAL1 UAS - HIS3 TATA - HIS3, MEL1 URA3:: GAL1UAS-GAL1 TATA - lacZ ).

Techniques:

Journal:

Article Title: Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase ?

doi:

Figure Lengend Snippet: Interactions between p167δ1 and p79 deletions. (A) The Y190 yeast strain was co-transformed with pACT-p167δ1 and pAS2-p79 and its deletions Δ1–Δ6. All p79 deletions were constructed in pAS2-1 vector as described in Materials and Methods. Colonies were picked and replica- plated on SD-Trp–/Leu– (a) and SD-Trp–/Leu–/His–+3AT plates (b), and incubated for 3 and 4 days, respectively. (a′) and (b′) represent results from the X-gal colony filter-lift assay for the respective plates. (B) β-Galactosidase activity was determined quantitatively using the liquid culture assay as described in Materials and Methods for the interaction between pACT-p167δ1 and pAS2-p79 and its deletions.

Article Snippet: The PCR-amplified DNA was cloned into pAS2-1 vector (Clontech Laboratories) at Nco I and Sal I sites, and the resultant plasmid pAS2p79 was transformed in the budding yeast strain, Y190 (in GAL4 2H-2; MAT a , ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4 Δ, gal80 Δ, cyhr2, LYS2::GAL1 UAS - HIS3 TATA - HIS3, MEL1 URA3:: GAL1UAS-GAL1 TATA - lacZ ).

Techniques: Transformation Assay, Construct, Plasmid Preparation, Incubation, Activity Assay

Journal:

Article Title: Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase ?

doi:

Figure Lengend Snippet: Interactions between p167δ2 and p79 deletions. (A) The Y190 yeast strain was co-transformed with pACT-p167 δ2, pAS2-p79 and its deletions Δ1–Δ6. All p79 deletions were constructed in pAS2 vector as described in Materials and Methods. Colonies were picked and replica- plated on SD-Trp–/Leu– (a) and SD-Trp–/Leu–/His–+3AT plates (b), and incubated for 3 and 4 days, respectively. (a′) and (b′) represent the results from the X-gal colony filter-lift assay for the respective plates. (B) β-Galactosidase activity was determined quantitatively using the liquid culture assay as described in Materials and Methods for the interaction between pACT-p167 δ2 and pAS2-p79 deletions.

Article Snippet: The PCR-amplified DNA was cloned into pAS2-1 vector (Clontech Laboratories) at Nco I and Sal I sites, and the resultant plasmid pAS2p79 was transformed in the budding yeast strain, Y190 (in GAL4 2H-2; MAT a , ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4 Δ, gal80 Δ, cyhr2, LYS2::GAL1 UAS - HIS3 TATA - HIS3, MEL1 URA3:: GAL1UAS-GAL1 TATA - lacZ ).

Techniques: Transformation Assay, Construct, Plasmid Preparation, Incubation, Activity Assay

Journal:

Article Title: Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase ?

doi:

Figure Lengend Snippet: Interaction of pACT-p167 deletions with p79. β-Galactosidase activities were determined quantitatively using the liquid culture assay as described in Materials and Methods for the interaction of pACT-p167 deletions δ1/δ2/δ3/δ4 with pAS2-p79 as well as with its deletion pAS2-p79 Δ1.

Article Snippet: The PCR-amplified DNA was cloned into pAS2-1 vector (Clontech Laboratories) at Nco I and Sal I sites, and the resultant plasmid pAS2p79 was transformed in the budding yeast strain, Y190 (in GAL4 2H-2; MAT a , ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4 Δ, gal80 Δ, cyhr2, LYS2::GAL1 UAS - HIS3 TATA - HIS3, MEL1 URA3:: GAL1UAS-GAL1 TATA - lacZ ).

Techniques:

Journal: Molecular Human Reproduction

Article Title: Isoform-specific GSK3A activity is negatively correlated with human sperm motility

doi: 10.1093/molehr/gaz009

Figure Lengend Snippet: GSK3 in human testis and spermatozoa and correlation with sperm motility. (A) Western blot analysis of total GSK3 and serine phosphorylated GSK3 isoforms in human testis and spermatozoa, mouse testis and HeLa cells; 30 μg of protein obtained in RIPA1x were loaded per sample. From top to bottom, GSK3 was immunodetected with the following antibodies: anti-GSK3A/B antibody, anti-GSK3A antibody and anti-GSK3B antibody, anti-GSK3A pS21 and anti-GSK3B pS9. (B) Immunoblot of total and serine phosphorylated GSK3 isoforms in human normospermic (n = 3) and asthenozoospermic (n = 4) ejaculated spermatozoa. (C) Total and serine phosphorylated GSK3 isoforms protein levels in human normospermic and asthenozoospermic (bar chart with error bars (SEM)). (D) Pearson Correlation between the percentage of progressive motile ejaculated sperm and protein levels of serine phosphorylated GSK3A and GSK3B (scatter plot with regression line). (E) Pearson Correlation between the percentage of progressive motile ejaculated sperm and protein levels of total GSK3A and GSK3B. Blots were cropped. *Correlation is statistically significant at the 0.05 level.

Article Snippet: The pAS2-1-GSK3A and pAS2-1-GSK3B vectors were transformed into AH109 yeast strain by a standard lithium acetate method (Clontech, Takara).

Techniques: Western Blot

Journal: Molecular Human Reproduction

Article Title: Isoform-specific GSK3A activity is negatively correlated with human sperm motility

doi: 10.1093/molehr/gaz009

Figure Lengend Snippet: Subcelular localization of GSK3A and GSK3B in normospermic ejaculated human sperm. GSK3A is located in the flagellum (star) and head (arrowhead), more specifically in the equatorial region. GSK3B is located through the entire head (arrowhead) and occasionally in the flagellum (star). Per sample, 100 sperm cells were counted. The experiment was done in triplicate. Scale bar is 5 μm. Nucleus is marked in blue. ROI: region of interest. All images were obtained with 63× magnification.

Article Snippet: The pAS2-1-GSK3A and pAS2-1-GSK3B vectors were transformed into AH109 yeast strain by a standard lithium acetate method (Clontech, Takara).

Techniques: