The membranes were placed in a shaker with the secondary antibody for 1 h at 20C, and subsequently washed 3 times with PBS. a shaker for 1 h at 20C. The membranes were subsequently washed three times using 1 ml PBS IL25 antibody for 5 min. The secondary antibodies horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG; cat. no. ab131368) and HRP-conjugated anti-rabbit IgG variable domain of heavy chain single domain (cat. no. ab191866) were added to the membranes. All primary and secondary antibodies were purchased from Abcam (Shanghai, China). The membranes were placed in a shaker with the secondary antibody for 1 h at 20C, and subsequently washed 3 times with PBS. Pierce? enhanced chemiluminescence western blotting substrate (Thermo Fisher Scientific, Inc.) was added to the membranes for 3 min, and the membranes were captured with the ChemiDoc XRS system (Bio-Rad Laboratories, Inc., Hercules, CA, Benzocaine USA). Immunofluorescence assay HCT-116 cells at the logarithmic growth phase were added to 6-well plates on a cover glass to form a control group (RPMI-1640, 10% FBS) and experimental groups with various concentrations of 17-AAG (1.25, 2.5 and 5 mg/l). The cells were collected after 48 h and washed once with PBS. Subsequently, 4% paraformaldehyde was added to the wells, and the cells were incubated at room temperature for 15 min prior to 3 washes with PBS. The cells were subsequently incubated with 1% Triton X-100 for 20 min at 20C and washed with PBS three times. Bovine serum albumin (1%; Beyotime Institute of Biotechnology) was added to the wells, which were then incubated for 30 min at room temperature. STAT3 primary antibody (1:200) was added to the wells and incubated overnight at 4C. The secondary antibody goat anti-mouse IgG (heavy chain and light chain; 1:400; cat. no. ab96879; Abcam) was added to the wells and incubated for 2 h at room temperature. The cells were washed three times with PBS. Following washing, DAPI was added to the wells and incubated for 5 min in the dark. The cells were observed Benzocaine under a fluorescence microscope and images were captured. Statistical analysis Statistical analysis was performed with SPSS (version 19.0; IBM SPSS, Armonk, NY, USA). The data were presented as the mean standard deviation. Data comparisons among groups were performed using one-way analysis of variance, and Turdey post hoc test. P 0.05 was considered to indicate a statistically significant difference. Results HCT-116 cell proliferation is inhibited by 17-AAG treatment The MTT assay results revealed that 1.25C20 mg/l of 17-AAG exhibited significant inhibitory effects (P 0.01) on the proliferation of HCT-116 cells in a concentration-dependent manner. The cell numbers in the 17-AAG treated groups were significantly reduced (P 0.01), compared with those observed in the control group, with an abnormal cell morphology exhibited by the 17-AAG-treated cells (Fig. 1). The proliferation inhibition rate of 17-AAG-treated cells (1.25, 2.5, 5, 10 and Benzocaine 20 mg/l) at 48 h (IC50, 1.71 mg/l) was increased, compared with that observed at 24 h (IC50, 23.24 mg/l; Table II; Fig. 2). Open in a separate window Figure 1. HCT-116 cells following culture for 48 h with various concentrations of 17-AAG; (A) control Benzocaine group; (B) 1.25 mg/l group; (C) 2.5 mg/l group; (D) 5 mg/l group. A decreased number of cells and abnormal cell morphology was observed in the 17-AAG treated groups, compared with the control. 17-AAG, 17-allylamino-17-demethoxygeldanamycin. Open in a separate window Figure 2. Inhibitory effects of 17-AAG-treatment on HCT-116 cells as assessed by flow cytometry. As the concentration of 17-AAG was increased, the inhibitory effect on the proliferation of HCT-116 cells also increased after 24 and 48 h. *P 0.01 compared with the control group. 17-AAG, 17-allylamino-17-demethoxygeldanamycin. Table II. Inhibitory effects of 17-AAG on the proliferation of HCT-116 colon carcinoma cells (mean standard deviation; n=6). is able to form apoptotic bodies with caspase regulatory factors, and activate caspase 9, and downstream caspase 3 and caspase 7 proteins, to initiate the process of cell apoptosis (34). Abnormal levels of apoptosis disrupt the balance between viable and dead cells to promote tumor development (35); therefore, the regulation of alterations in apoptosis may be a novel anticancer therapy. This present study identified the apoptosis-inducing ability of 17-AAG, but the underlying.