Anti trim14

The cells or tissues were lysed, and the total protein concentration was measured using the BCA assay. Equal quantities of proteins were resolved using SDS-PAGE and then transferred onto a PVDF membrane (Merck Millipore, Billerica, MA, USA). Next, the membrane was blocked at 37 °C for 2 h and then incubated with the primary antibody (anti-COLII, anti-ACAN, anti-NF-κB, anti-IFN-β, anti-GAPDH [all from Abcam, Cambridge, MA, USA], or anti-Trim14 [Novus Biologicals] antibody) at 4 °C overnight. The membrane was washed three times with PBST (PBS with Tween 20, Solarbio) and then incubated with the horseradish-peroxidase-labeled anti-mouse or anti-rabbit IgG secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) (diluted 1: 10,000 with PBST) at 37 °C for 2 h. Finally, the membrane was washed five times with PBST and developed using chemiluminescence (BeyoECL Star [Beyotime, Shanghai, China]). The results were analyzed using the ImageJ software (NIH, Bethesda, MA, USA).

To evaluate the therapeutic potential of EVs-150 in OA, IF colocalization analysis was performed to observe the extent of the AC injury and repair and the changes in the related signaling pathways. After antigen retrieval, tissue sections were incubated with the anti-COLII, anti-ACAN, anti–NF-κB (all from Abcam, Cambridge, MA, USA), and anti-Trim14 (Novus Biologicals, Littleton, CO, USA) antibodies as the primary antibodies and then with the Cy3- or Alexa fluor-488-labeled secondary antibody (Abcam, Cambridge, MA, USA) for fluorescent detection. The cell nucleus was stained with DAPI. The tissue sections were immediately examined and photographed under an inverted fluorescent microscope (IX-73, Olympus, Tokyo, Japan) to determine the fluorescence intensity and localization.