Journal: bioRxiv

Article Title: Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue

doi: 10.1101/2024.01.15.575715

Figure Lengend Snippet: Assessing the top p-S65-Ub antibodies in ELISA using recombinant protein. ( A ) Sandwich ELISA results for the detection of recombinant K48 (solid line) and K63 (dash line) p-S65-Ub tetramers (Ub4) that were serially diluted to the range of 1–1,000,000 pg/ml. ( B ) Direct ELISA results for the detection of p-S65-PRKN and nonphosphorylated PRKN recombinant proteins that were serially diluted to the range of 1–1,000,000 pg/ml. N = 2. Data are shown as mean with SD.

Article Snippet: Recombinant p-S65-PRKN (Boston Biochem, E3–166), non-phosphorylated PRKN (Boston Biochem, E3–160) and p-S65-Ub monomer (Boston Biochem, U102) were initially dissolved in RIPA buffer (stock concentration of 1 μg/μl), aliquoted in 1 μl aliquots, flash-frozen in liquid nitrogen and stored at −80°C.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Sandwich ELISA, Direct ELISA

Journal: Biology of reproduction

Article Title: Antiproliferative effects of adiponectin on human trophoblastic cell lines JEG-3 and BeWo.

doi: 10.1095/biolreprod.108.070573

Figure Lengend Snippet: FIG. 2. Phosphorylation of (P-PRKA) by adiponectin in BeWo and JEG-3 cells. Effects of adiponectin on PRKA activation in BeWo (A and C) and JEG-3 (B and D) cells. Cells were maintained in a serum-free culture medium overnight. Cells were then exposed to human recombinant adiponectin (250 ng/ml) or AICAR (500 lg/ml). At the indicated times, cellular extracts were prepared and immunoblotted with anti-phospho-PRKA or with anti-total PRKA. A and B) Western blot analysis from a representative experiment. C and D) Densitometric analysis of PRKA immunoblots. The figure shows means of active phospho-PRKA/total PRKA ratios. Results are means 6 SEM obtained from four to six separate experiments and are expressed as percentages of control value (untreated). *P , 0.05, Wilcoxon test; ns, nonsignificant.

Article Snippet: Proteins were transferred to PVDF membrane and were blocked in buffer A with 2.5% gelatin during 2 h. Membranes were incubated overnight at room temperature in buffer A with 2.5% gelatin rabbit polyclonal anti-phospho-PRKA antibody (1:700 dilution, #2531; Cell Signaling Technology, St-Quentin-en-Yvelines, France) or rabbit polyclonal anti-total PRKA antibody (phosphorylated and nonphosphorylated forms) (1:1000 dilution, #2532; Cell Signaling Technology).

Techniques: Phospho-proteomics, Activation Assay, Recombinant, Western Blot, Control