Bs 0295 hrp

The brains were quickly extracted and LHb tissues were removed bilaterally. The tissue was collected and homogenized in RIPA lysis buffer containing 12 μg phenylmethylsulfonyl fluoride. Proteins were loaded onto 12% SDS-PAGE gels, electrophoretically separated, and transferred to polyvinylidene fluoride membranes for western blot analysis. The primary antibodies used were β calmodulin-dependent protein kinase type II (βCaMKII, ab34703; Abcam, Cambridge, UK) and anti-β-actin (bs-0061-R; Bioss Inc., Woburn, MA, USA). These antibodies were detected using horseradish peroxidase-conjugated goat anti-rabbit antibody (bs-0295-HRP; Bioss Inc., Woburn, MA, USA). High sensitivity electrochemiluminescence reagent was used to visualize the bands.

After washing in ice-cold PBS, the acquired tissues and cells were processed with ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer complemented with phenylmethylsulfonyl fluoride (PMSF, at 1 mM), followed by Western blotting in accordance with the previously mentioned methods [28 (link)], with Occludin (diluted at 1 : 1000; bs-10011R; Bioss), ZO-1 (dilution rate 1 : 1000; bs-1329R; Bioss), p-AMPK (Ser485) (diluted at 1 : 1000; #2537; Cell Signaling Technology; Danvers; USA), Claudin-11 (dilution rate 1 : 1000; bs-21509R; Bioss), AMPK (Ser485) (1 : 1000 dilution; #2532; Cell Signaling Technology), SIRT3 (diluted at 1 : 1000; 10099-1-AP; Proteintech), β-actin (1 : 3,000 dilution; bs-0061R; Bioss), SOD2 (dilution rate 1 : 1000; bs-23402R; Bioss), NLRP3 (1 : 1000 dilution; 19771-1-AP; Proteintech), and IL-1β (dilution rate 1 : 1000; #12703; Cell Signaling Technology) as the primary antibodies, as well as the HRP-labeled goat anti-rabbit antibody (diluted at 1 : 3000, bs-0295-HRP; Bioss) as the secondary antibody. Ultimately, the ECL solution was applied to measure the bands, and the Image J software was employed to determine the signals.