Western Blot Analysis of Protein Targets

After isolating total proteins from HESCs or nucleus with the lysis buffer, a BCA kit (Abcam, Cambridge, UK) was utilized to determine the concentration of proteins, followed by loading the proteins into the 12% SDS-PAGE. After separation, proteins in the gel were transferred onto the PVDF membrane (Abcam, Cambridge, UK), followed by incubation in the TBST buffer containing the primary antibody against COX-2 (1:1000, GeneTex, Texas, USA), p-p38 (1:2000, GeneTex, Texas, USA), p-IκBα (1:500, GeneTex, Texas, USA), IκBα (1:3000, GeneTex, Texas, USA), NF-κB p65 (1:3000, GeneTex, Texas, USA), or Tubulin (1:8000, GeneTex, Texas, USA). After incubating at 4°C overnight, the membrane was incubated with the secondary antibody (1:2000, GeneTex, Texas, USA) at room temperature for 90 min. Lastly, the ECL solution was utilized to expose the membrane and the expression of proteins was quantified using the Image J software [13 (link)].