Af5142

Western blotting was performed to assess the influence of the
CAV1-YAP-mediated mechanotransduction pathway and the anti-inflammatory
effects of the NPC-encapsulated composite hydrogel under inflammatory
conditions. Proteins were labeled and incubated overnight at 4°C with the
following primary antibodies: YAP (1:500, A1001, ABclonal), p-YAP (1:500,
AP0489, ABclonal), CAV1 (1:2000, 16447-1-AP, Proteintech), Col II (1:2000,
ab188570, Abcam), Agg (1:2000, DF7561, Affinity), IL-1β (1:1000, AF5103,
Affinity), IL-6 (1:1000, DF6087, Affinity), IL-10 (1:1000, DF6894,
Affinity), IL-4 (1:1000, AF5142, Affinity), and β-actin (1:5000, AF7018,
Affinity). After washing using TBST buffer, the membranes were treated with
horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, S0001,
Affinity) for 2 h at room temperature. Then examined with a chemiluminescent
reagent (Beyotime), and quantified the signal intensity using ImageJ
software.

To evaluate in vivo changes in ECM secretion, levels of
inflammatory cytokines, and macrophage polarization, the sections were
immunohistochemically stained. Briefly, deparaffinized sections were
incubated with 3% H₂O₂ for 15 min in the dark and then
blocked with 3% BSA for 30 min at room temperature. The sections were
incubated overnight at 4°C along with the following primary antibodies: Col
II (1:500, GB111629, Servicebio), aggrecan (1:500, GB11373, Servicebio),
IL-1β (1:200, AF5103, Affinity), IL-6 (1:200, DF6087, Affinity), IL-10
(1:200, DF6894, Affinity), IL-4 (1:200, AF5142, Affinity), CD68 (1:300,
GB113109, Servicebio), Arg1 (1:500, GB11285, Servicebio), and CC-chemokine
receptor 7 (CCR7) (1:300, GB11502, Servicebio). After primary antibody
incubation, the sections were incubated with a specific HRP-conjugated
secondary antibody (1:200, S0001, Affinity) for 1 h at 25°C. The sections
were stained with 3,3′-diaminobenzidine and counterstained with hematoxylin
for the nucleus. Finally, the sections were mounted and photographed using
an optical microscope (Nikon, Tokyo, Japan). Immunohistochemical staining
intensity was digitally quantified using ImageJ software.