Journal:

Article Title: Translesion Synthesis past Equine Estrogen-derived 2′-Deoxyadenosine DNA adducts by Human DNA polymerases η and κ

doi: 10.1021/bi0525324

Figure Lengend Snippet: Two hundred μg of 5′TTTGTATTTT was reacted at 37 °C for 16 h with 4-OHEN (1 mg/50 μL acetone) in 1 mL of 25 mM potassium phosphate buffer, pH 7.4. After centrifugation, the supernatant was evaporated in dryness and subjected to HPLC. The 10-mer oligomers containing an isoform of 4-OHEN-dA adduct (5′TTTGTA4-OHENTTTT) were isolated on a reverse-phase μBondapak C18 column (0.39 × 30 cm, Waters), using a linear gradient composed of 0.05 M triethylammonium acetate (pH 7.0) containing 10 → 30 % acetonitrile, an elution time of 60 min, and flow rate of 1.0 mL/min.

Article Snippet: HPLC grade acetonitrile and triethylamine were purchased from Fisher Chemical.

Techniques: Centrifugation, Isolation

Journal:

Article Title: Translesion Synthesis past Equine Estrogen-derived 2′-Deoxyadenosine DNA adducts by Human DNA polymerases η and κ

doi: 10.1021/bi0525324

Figure Lengend Snippet: 4-OHEN-dA-modified oligodeoxynucleotides [5′TTTGTA4-OHENTTTT, 1.5 μg for Pk-2 (B) and Pk-3 (C)] was digested at 37 °C overnight with micrococcal nuclease (30 units) and spleen phoshodiesterase (0.15 units) and was further incubated at 37 °C for 2 h with alkaline phosphatase (3 units) in a buffer, as described in Materials and Methods. The resulting deoxynucleosides were analyzed using HPLC, using a μBondapak C18 column (0.78 × 30 cm, Waters). Elution was carried out using a linear gradient composed of distilled water containing 10 % methanol over 2 min, 10 - 37 % methanol over 5 min, and 37 - 90 % methanol over 19 min at a flow rate of 2.0 mL/min. (A), a mixture of dNs and four diastereoisomers of 4-OHEN-dA. Diastereoisomers (fr-1, fr-2, fr-3, and fr-4) of monomeric 4-OHEN-dA were prepared as described previously (11).

Article Snippet: HPLC grade acetonitrile and triethylamine were purchased from Fisher Chemical.

Techniques: Modification, Incubation

Journal: bioRxiv

Article Title: Nicotinamide riboside activates renal metabolism and protects the kidney in a model of Alport syndrome

doi: 10.1101/2024.02.26.580911

Figure Lengend Snippet: ( A ) Representative images of immunohistochemistry for p57 kip2 , followed by periodic acid–Schiff post-staining without hematoxylin counterstaining. Podocyte nuclei are stained brown, and all other glomerular structures are stained pink. ( B ) Quantification of p57 kip2 immunostaining with PodoCount, a validated algorithm to analyze p57 kip2 -stained whole slide images. Podocyte volumetric density was reduced in Alport mice and restored by NR treatment in both sexes. ( C,D ) Immunoblots for KIM-1, a tubular injury marker, in male ( C ) and female ( D ) kidney homogenate. Ponceau S, a nonspecific protein stain, was used as a loading control. ( E,F ) Quantification of immunoblots ( C,D ) shows that KIM-1 is increased in Alport mice and reduced by NR treatment in male ( E ) and female ( F ) mice. Scale bars represent 100 µm. Significance was determined by one-way ANOVA with the Holm–Šídák correction for multiple comparisons. Data are expressed as the means ± SEM. Each datum represents one glomeruli ( B ) or one mouse ( E-F ). * P < 0.05, ** P < 0.01, **** P < 0.0001. Kidney injury molecule-1, KIM-1; NR, nicotinamide riboside; PSR, picrosirius red; Veh, vehicle.

Article Snippet: Immunohistochemistry for p57 kip2 (Cat. No. ab75975, Abcam) followed by periodic acid–Schiff (Cat. No. 22-110-645, Fisher Scientific, Hampton, NH) post-staining without hematoxylin counterstaining was performed and analyzed as previously described ( ).

Techniques: Immunohistochemistry, Staining, Immunostaining, Western Blot, Marker