Anti eno1

Monoclonal antibodies; including rabbit anti-PCK1 (#12940), rabbit anti-PCK2 (#8586), rabbit anti-c-Myc (#13987) and the polyclonal rabbit anti-PARP (#9542) were purchased from Cell Signaling Technology (Danvers, MA). The monoclonal mouse anti-His-tag (#ab18184), anti-β-actin (#ab8229), anti-G-6-Pase (#ab83690) and anti-ENO1 (#ab112994) were purchased from Abcam (Cambridge, UK). The polyclonal rabbit anti-Bcl-xL (#AB126) and the polyclonal rabbit anti-rabbit AKT (#AA326) were obtained from Beyotime (Shanghai, China). The monoclonal mouse anti-β-tubulin (#CW0098M) and fluorescein (FITC)-conjugated affinipure goat anti-mouse IgG antibody (#121217) were products of ComWin Biotech (Beijing, China) and Jackson ImmunoResearch Laboratories (West Grove, PA) respectively.

The human SCLC cell lines H69, H446 and H69AR were purchased from American Type Culture Collection (ATCC, USA). H446DDP was constructed by exposing H446 cells to cisplatin for 6 months in our laboratory. The IC50 values of these four cells are shown in Figure S1A. H69AR and H446DDP were maintained in drug‐free medium for at least 2 weeks before experiments. All cells were cultured in RPMI‐1640 medium (Hyclone) containing 10% foetal bovine serum (Gibco) and antibiotics (100 mg/mL penicillin and 100 mg/mL streptomycin).
ENOblock and LY294002 were purchased from Selleck Chemicals. To inhibit the function of ENO1 or the activities of the PI3K/Akt pathway, cells were treated with 10 μmol/L ENOblock or 12 μmol/L LY294002 for 48 hours. The following antibodies were used: anti‐FGFRL1, anti‐HuR and anti‐ENO1 were purchased from Abcam; anti‐BAX, anti‐BCL2 and anti‐p‐Akt 427 were from Proteintech; anti‐PARP, anti‐p‐PI3K and anti‐p‐Akt 308 were from Affinity; anti‐T‐Akt and anti‐T‐PI3K were from Wanlei; and anti‐GAPDH was from Bioworld.

WT and IL-10^−/− DCs (1 × 10⁶ cells/ml) were pulsed with Chlamydia (MOI of 5) for 1 and 2 h, and then fixed for 5 min at room temperature in PBS solution containing 4% formaldehyde/ 0.01% glutaraldehyde. Samples were washed twice in cold 1X PBS and the Fc receptors were blocked with Fc blocker. DCs were incubated with Anti-ENO1 (Abcam, Cambridge, MA) for 1 h at 4 °C and washed twice with 1X PBS. DRAQ5 and Alexa Fluor 488 bound secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were then added for 1 h at 4 °C. DCs were washed three times and resuspended in wash buffer overnight at 4 °C. Confocal images were obtained on the Zeiss 510 VIS 234 Confocal Microscope (Carl Zeiss Microscopy, GmbH). Images were taken from different fields on each plate. Quantitative colocalization analysis was used in analyzing the data (ImageJ Software, NIH, USA). We repeated this experiment 3 times.

To determine the expression level of ENO1 protein in CFPAC‐1 and MiaPaCa‐2 cells, the cells (1 × 10⁷) were harvested for Western blot analysis. In addition, 20 μg of total protein was separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), followed by transferring into nitrocellulose filter (NC) membranes (EMD Millipore) using a semi‐dry electrophoretic transfer cell system (Bio‐Rad Laboratories, Inc). The membranes were blocked with 5% non‐fat milk for 1 hour and then incubated with anti‐ENO1 (Abcam) or an antibody specific to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (Sigma‐Aldrich Corp.) overnight at 4°C. The membranes were then incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies (Santa Cruz Biotechnology, Inc) for 1 hour at room temperature. Blots were detected with enhanced chemiluminescence (ECL) reagents (EMD Millipore) and exposed to a chemiluminescent imaging system for 5 minutes.