Ab129200

Eight paraffin blocks showing continuous histopathological transition from distant endometriosis, contiguous atypical endometriosis and ovarian clear cell carcinomas were selected for sectioning. Five of the blocks were genotyped as carrying the C/C genotype, three as carrying T/T genotype at rs11614913 in MIR196A2, and were utilized for this study. Immunofluorescence staining was performed to detect active cell nucleoli and ribosome biogenesis activity using 1:100 rabbit anti-nucleophosmin (anti-NPM; ab52644) and anti-nucleolin (anti-NCL; ab129200) monoclonal antibodies (Abcam PLC, Cambridge, MA). Immunostaining was independently scored by two pathologists, and specific nucleolus staining was scored as: negative (0), weakly positive (1+), moderately positive (2+) or strongly positive (3+). We used a combination of the percentage of positively stained cells and the intensity of nucleolus staining for statistical analysis. The H-score = ΣPi xi was calculated, where i is the intensity of the stained tumor cells (0 to 3+) and Pi is the percentage of the stained tumor cells for each intensity group (0 to 100%) as previously described [67 (link)]. For discordant cases, a third investigator was brought in to score and the final intensity score was determined by the majority scores.

HEK293T effector cells were plated in a 12 well-plate in presence of PHI and doxycycline. The following day, they were stained with an APC conjugated anti-RSV-F antibody and with LIVE/Dead™ Fixable Aqua (Life Technologies) for 30 min at 4°C. Cells were analysed on a MACSQuant Analyzer flow cytometer (Miltenyi Biotec) and data analyzed using FlowJo (TreeStar). HEp-2 or Calu-3 cells were seeded in a 6 well-plate and incubated with Daprodustat (50 mM or 100 mM) for 48h. Cells were trypsinised, stained with LIVE/Dead™ Fixable Aqua (Life Technologies), permeabilized for 15min at room temperature (0.1% Triton X-100 in PBS), fixed with 4% paraformaldehyde (PFA, Sigma) for 10min at room temperature and blocked for 20min on ice (0.5% BSA, 0.5% tween in PBS). Cells were stained in darkness with primary nucleolin antibody (abcam, ab129200) for 20 min on ice, followed by staining with in fluorescent dye conjugated secondary antibody (goat anti-rabbit Alexa 488nm, Thermo Fisher, A11034) for 20min on ice. Data was acquired using an Attune NxT flow cytometer (Thermo Fisher) and analyzed using FlowJo (TreeStar).

Anti-β-actin (M1210-2) mouse monoclonal antibody (mAb) and anti-Myc (R1208-1), anti-FLAG (0912-1), and anti-GFP (SR48-02) rabbit polyclonal antibodies (pAbs) were purchased from Huaan Biological Technology (Hangzhou, China). Anti-GFP (B-2, sc-9996) mouse mAb for immunoprecipitation was acquired from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-FLAG (F1804), and anti-Myc (05-419) mouse mAbs used for IP were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-FLAG affinity resin (A2220) for immunoprecipitation (IP) was purchased from Sigma-Aldrich. Rabbit mAb against YTHDC1 (ab259990) and rabbit mAb against NCL (ab129200) were purchased from Abcam (Cambridge, MA, USA). Anti-PEDV N mAb was prepared and stored in our laboratory. NP-40 cell lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, and 1% NP-40) was obtained from Beyotime (P0013F; Shanghai, China). Horseradish peroxidase (HRP)-labeled goat anti-mouse and anti-rabbit IgG antibodies were purchased from KPL (Milford, MA, USA).