Fitc goat anti mouse igg

The antibodies used in the present study were purchased from Cell Signaling Technology, including Bcl-2, Bax, caspase 3, β-actin, LC3, AKT, phospho-AKT (Ser473), mTOR, phospho-mTOR (Ser2448), S6 ribosomal protein, phospho-S6 ribosomal protein (Ser235/236) and ATG5, with their catalog number D17C4, D2E11, D3R6Y, E4D9Z, D11, 11E7, D9E, 7C10, D9C2, 54D2, D57.2.2E, and D5F5U, respectively. Other antibodies included cytochrome c (M1701-9), goat anti-rabbit/mouse IgG-HRP (ImmunoResearch Laboratories) and FITC goat anti-mouse IgG (Beyotime, A0562).
Reagents used in our research included: cell cycle and cell apoptosis analysis kits (KEYGEN, KGA511 and KGA107); Hochest33342 staining kit (Beyotime, C1025); mitochondrial membrane potential detection kit and ROS measurement kit (NJJCBIO, G009-1-3 and E004-1-1), MitoTracker Deep Red FM (Life Technologies, M22426), Lyso-Tracker Red (Invitrogen, L7528), cholorquine (CQ, PubChem, 2719), 3-MA, and N-acetylcysteine (NAC) (aladdin, M129496 and A105422). Moracin N (MAN) was isolated and purified by Prof. Tian Jingkui from the Key Laboratory of Biomedical Engineering at Zhejiang University.

BT474 and SKBR3 cells were induced with JAC1 or vehicle (DMSO) for 24 h. Subsequently, the cells were fixed with methanol for 30 min, washed with PBST, and 10% normal goat serum for 1 h aiming to block non-specific signals. The cells were incubated with anti-JWA antibody (1:200) and anti-HER2 (1:250) overnight at 4 °C. After washing with PBST, the FITC goat anti-mouse IgG and CY3 goat anti-rabbit IgG (1:100, Beyotime, Jiangsu, China) were used to incubate with cells for 2 h. After washing three times with PBST, the cells were stained with DAPI (Beyotime, Jiangsu, China) for 20 min. The confocal images of the cells were captured using Zeiss AIM software on a Zeiss LSM 700 confocal microscope system (Carl Zeiss Jena, Oberkochen, Germany).