Ecl system

Human pancreatic tumor cells or pancreatic tumor tissues were lysed in RIPA lysis buffer with 1% deoxycholate, 1.0% Triton X-100, and 0.1% SDS in 0.01 M Tris base (pH 7.4), 0.15 M NaCl, and containing the following inhibitors, 100 μM phenylmethanesulfonyl fluoride (PMSF), 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 100 μM sodium vanadate, essentially as described previously53 (link). Protein concentration of whole cell or tissue lysate was determined by BCA kit (Pierce, Rockford, IL). Equivalent micrograms of whole cell or tissue lysates were electrophoresed on SDS PAGE, transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA), probed with indicated antibodies, and developed with ECL system (Pharmacia Biotech, Piscataway, NJ) as described56 (link). Densitometry analysis of band density was described previously53 (link). The densitometric readings were pooled and averaged from three independent experiments. Density was relative to GAPDH and the relative highest band density was as 100%. The background of densitometric reading on the ECL-developed film was subtracted. The expression of GAPDH protein was used as a loading control.

Protein production was determined by Western blot analysis as described by us previously.23 Cells or tissues were lysed in 1% NP‐40 lysis buffer containing the following inhibitors, 100 μmol/L PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 100 μmol/L sodium vanadate and 20 μg/mL TLCK. Protein concentration of whole tissue or cell lysate was determined by BCA kit (Pierce). Equivalent micrograms of lysates were electrophoresed on SDS‐PAGE, transferred to Immobilon‐P membrane (Millipore Corp.), probed with indicated antibodies and developed with ECL system (Pharmacia Biotech). The beta‐actin protein level was used as a loading control. Quantitative analysis (densitometry) of Western blots was performed by calculating the relative density (pixel density) of the immunoreactive bands after acquisition of the blot image (scanning) and analysis with Adobe Photoshop software as described.24 The background of densitometric reading on the ECL‐developed film was subtracted.

Western blot assays were performed as described by us previously [36 (link)]. Briefly, equivalent micrograms of whole cell lysates were electrophoresed on a disulfide-reduced 7.5% SDS-PAGE, transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA, USA), probed with indicated antibodies and developed with ECL system (Pharmacia Biotech, Piscataway, NJ, USA). The expression of G3PDH protein was used as a loading control. For densitometric analysis of band intensity, a specific band on the enhanced chemiluminescence (ECL)-developed film was subjected to densitometric analysis (Adobe Photoshop). The densitometric readings were pooled and averaged from three independent experiments as described by us [35 (link)]. The background of densitometric reading on the ECL-developed film was subtracted.

H9 cells cultured overnight in RPMI1640 medium/0.1% BSA were treated with CCL19 or CCL21 at 37 °C, and then washed with media containing ice-cold PhosStop (Roche, Basal, Switzerland). The cells were lysed by adding 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton-X, PhosStop and Protease Inhibitor Tablets (Sigma). The samples were separated by SDS-PAGE, and transferred onto PVDF membranes for immunoblotting with an anti-phospho-Akt, phospho-Erk1/2, total-Akt, total-Erk1/2 antibody (Cell Signaling Inc., MA, USA) or anti-GAPDH antibody (Biolegend). Bound antibodies were detected using the ECL system (GE Healthcare Life Sciences).

Total protein was extracted from lung tissues or RAW264.7 cells using RIPA lysis buffers containing protease inhibitors and phosphatase inhibitors. The concentrations of proteins were measured using the BCA protein quantification kit (Thermo Fisher Scientific, Waltham, USA). Equal amount of protein samples was boiled and loaded in 10% SDS-PAGE for separation and transferred to PVDF membrane (Meck Millipore, Burlington, USA). The PVDF membranes were blocked with 5% skim milk at room temperature for 1 hour and then incubated with different primary antibodies at 4°C overnight. The membranes were incubated with the corresponding secondary antibodies for 1 h at room temperature. The proteins were visualized by chemiluminescence using an ECL system (GE Healthcare, Pittsburgh, USA) and the images were captured using an imaging system (Tanon, Shanghai, China).

Whole cell lysates from the cultured HEK293 cells and normal placental tissue (total 1 mg of protein) were incubated with VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. Following incubation, immunocomplexes were immunoprecipitated using protein A/G agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C with gentle rotation. The beads were washed three times with lysis buffer and boiled in 50 μl of 1X SDS sample buffer for 5 min at 95 °C. After centrifugation, the precipitated proteins were separated by SDS-PAGE and electrophoretically transferred onto an enhanced chemiluminescence (ECL) nitrocellulose membrane (GE Healthcare, London, UK). Western blot analysis was performed with the indicated primary antibodies followed by appropriate horseradish peroxidase–conjugated secondary antibodies using an ECL system (GE Healthcare, Buckinghamshire, UK), as described previously^{45 (link)}. Equal protein loading was confirmed by Ponceau S staining and sequential incubation of the membrane with anti-β-actin (Sigma-Aldrich) antibody.