West q chemiluminescent substrate kit

HuCCT1 or H69 cells treated with 800 ng/mL ESPs, for the indicated times, were washed with ice-cold PBS and then lysed with a RIPA buffer containing a complete protease inhibitor cocktail (Sigma-Aldrich). Thirty μg of total soluble protein was separated by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane (Millipore, Bedford, MA). Membranes were probed with primary antibodies against E-cadherin (1:3000 dilution) or N-cadherin (1:1000). After incubation with host-specific secondary antibodies, the immunoreaction was detected with a West-Q-chemiluminescent substrate kit (GenDEPOT, Barker, TX) and quantitated by densitometric scanning of the X-ray film with a Fluor-S Multimager (Bio-rad, Hercules, CA). The blots were normalized for protein loading by washing in Blot-Fresh Western Blot Stripping Reagent (SignaGen Laboratories, Gaithersburg, MD) and re-probing with a polyclonal antibody against GAPDH (1:5000 dilution).

Cells were washed with ice-cold PBS and lysed with RIPA buffer (Sigma-Aldrich) supplemented with complete protease inhibitor cocktail and centrifuged at 13,000× g at 4 °C for 20 min. Nuclear proteins were isolated using the NE-PER Cytoplasmic and Nuclear Protein extraction kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. Protein concentration was determined with the BCA Protein Assay kit (Pierce Biotechnology). Next, 30 μg of total soluble proteins or 5 μg of nuclear proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Merck-Millipore, Billerica, MA, USA). The membranes were probed with primary antibodies (1:1000 dilution) described in Materials, followed by incubation with appropriate secondary antibodies (1:5000 dilution). The immunoreactive bands were visualized with a West-Q chemiluminescent Substrate kit (GenDEPOT, Barker, TX, USA) and the band intensities on films were analyzed by densitometry to quantify protein expression using a FluorS MultiImager (Bio-Rad, Hercules, CA, USA). The membranes then were washed with Restore Western Blot Stripping Buffer (Thermo Scientitis, Waltham, MA, USA) and reprobed with 1:1000 diluted anti-GAPDH polyclonal or anti-Lamin B polyclonal antibodies to normalize for cytosolic or nuclear protein loading, respectively.

Briefly, the five ovalbumin-conjugated peptides were separated by SDS-PAGE and stained with Coomassie Blue. Separated peptide fractions were electroblotted onto Immobilon-P Transfer membranes (Millipore, USA), which were then blocked with 5% skim milk (Wako, Japan). Subsequently, membranes were probed overnight with vivax malaria sera samples diluted in 5% skim milk. Bound antibodies were reacted with horseradish peroxidase-conjugated secondary antibodies and detected using the West-Q Chemiluminescent Substrate Kit (GenDEPOT, USA).