Ab235585

Structural remodeling occurs after MI, which can lead to changes in gap junctions among cardiac myocytes. In the previous study, we observed CCR9 knockout could improve these serious structural remodeling, so we wondered whether CCR9 knockout could affect these changes in gap junction. Here, western blot was applied to detect the expression level of connexin 43 (Cx43) in ventricular tissue from infracted border zone. The mice were sacrificed to dissect the LV infract border zone. The dissected tissues were grinned to extracted proteins and quantified protein concentrations by BCA assay kit. Forty microgram protein sample was loaded and proteins were separated by 10% sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis (PAGE), then transferred to a polyvinylidene difluoride (PVDF) membrane. After the transferring, the PVDF membrane was removed and transferred into sealing solution for mild vibration at 4°C overnight with anti-Cx43 primary antibody (1:1,000, abcam, ab235585) followed by rabbit IgG (1:2,000, abcam, ab6721) for 1 h at room temperature. Development and photographic recording were performed with Bio-RDA gel imaging system.

Cells were lysed with precooled RIPA lysis and 15‐min centrifugation (at 14,000g and 4°C) was performed. Then the supernatant was removed to a new tube. Anti‐STIP1 (ab126753, 1:20, Abcam), anti‐Cx43 (ab235585, 1:30, Abcam), anti‐HSP70 (ab2787, 1:50, Abcam), or anti‐HSP90 (ab59459, 1:50, Abcam) was added to 1 mL cell lysate, and IgG antibody (ab172730, 1:100, Abcam) was added to the NC group. The antigen‐antibody mixture was slowly shaken on a shaker overnight at 4°C or for 2 h at room temperature. After that, the mixture was added with 100 μL Protein A/G agarose beads (prepared in PBS with a concentration of 50%) and incubated at 4°C overnight or for 1 h at room temperature. The agarose bead‐antigen‐antibody complex was obtained after centrifugation at 14,000 rpm for 5 s. Next, the complex was rinsed three times with precooled RIPA (800 μL) and suspended with 2× loading buffer (60 μL). The sample was boiled for 5 min and centrifuged to collect the remaining agarose beads, and the supernatant was boiled again for 5 min and then electrophoresed. Protein expression was measured by western blotting.