Goat anti mouse igg h l hrp

The tissues were homogenized in a RIPA (P0013C) lysis buffer containing 1 mM PMSF (ST507). A BCA kit (P0012) was used to measure the concentration of the total protein. In this study, 20–30 ug of proteins from each sample was loaded onto a sodium dodecyl sulfate (SDS) gel for electrophoresis and then transferred to polyvinylidene difluoride (PVDF, 03010040001, Sigma, St. Louis, MI, USA) membranes. Subsequently, the blots were incubated with the primary antibodies overnight at 4 °C. On the second day, the membrane was washed and then reacted with the secondary antibody for 1 h at room temperature. Finally, the blots were visualized with an enhanced luminol-based chemiluminescent kit (34075, Thermo Fisher, Waltham, MA, USA) and scanned by an electronic camera system. The primary antibodies rabbit anti-β-actin (1:1000, 4970S), rabbit anti-IL-6 (1:1000, 12912T), rabbit anti-IL-1β (1:1000, 12703S), and rabbit anti-pP65 (1:1000, 3033S) were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary rabbit antibody P65 (1:1000, Ab16502) was purchased from Abcam (Cambridge, UK). The secondary antibodies goat anti-rabbit IgG (H+L) HRP (1:5000, A0218) and goat anti-mouse IgG (H+L) HRP (1:5000, A0216), RIPA, PMSF, and the BCA kit were purchased from Beyotime (Shanghai, China).

RIPA lysis buffer (Thermo Fisher) was applied to extract proteins from cells. Western blot assay was performed with reference to previous report (He et al., 2021 (link)). The antibodies used were: PDK4 (1:7000; Proteintech, 12949-1-AP), GAPDH (1:15000; Proteintech, 60004-1-Ig), Goat Anti-Mouse IgG H&L(HRP) (1:1000; Beyotime, A0216), Goat Anti-Rabbit IgG H&L(HRP) (1:1000; Beyotime, A208). Western blots were detected by ECL luminescence kit (Thermo Fisher) and visualized by chemiluminescence apparatus. ImageJ software was used for quantification.

RIPA lysis buffer (Thermo) was utilized to extract total protein from exosomes and HUVECs. BCA kit (Thermo Scientific, USA) was applied to determine total protein concentration. We used 10% SDS-PAGE to separate the samples, and then transferred samples to PVDF membranes. Subsequently, we utilized 5% milk to block the membranes at 24 °C temperature and treated with anti-CD63 primary antibodies (1:1000; Abcam, ab216130), CD9 (1:1000; Proteintech, 20597-I-AP), LFNG (1:5000; Abcam, ab151699), and β-actin (1:10,000; Proteintech, 60008-I-Ig) were incubated overnight at 4 °C. After washing 3 times with TBST buffer, the goat antirabbit IgG H&L (HRP) (1:1000; Beyotime, A0208) and goat antimouse IgG H&L (HRP) (1:1000; Beyotime, A0216) were incubated for 2 h at room temperature. Next, the high sensitive ECL luminescence kit was used for color development, the chemiluminescence imaging analysis system was exposed, and the pictures were collected. Finally, Image J (v5.2.1) was used for densitometric analysis.