After the incubation was complete, bacteria were pelleted via centrifugation at 18,900 × g and the supernatants were solublized by boiling in 2× SDS-PAGE sample buffer containing 2-mercaptoethanol. Samples were subjected to 10% SDS-PAGE and then SN-38 electrophoretically transferred to a PVDF membrane (Immobilon-P, Millipore). The PVDF membrane was pre-blocked with 1% BSA-TBST for 1 hour at RT to minimize non-specific protein binding, and was then incubated with sheep anti-human fibronectin-specific antibody (diluted 1:2000 in 1% BSA-TBST) for 1 hour at RT with

gentle rocking. The PVDF membrane was washed three times with TBST to remove unbound primary antibody. The membrane was then incubated in a solution of anti-sheep/goat IgG monoclonal antibody (GT-34, diluted 1:5000 in 1%BSA-TBST) with rocking eFT-508 concentration A 769662 for 1 hr at RT. The PVDF membranes were washed 3 times with TBST to remove unbound secondary antibody. The blot was developed using Pierce PicoWest chemiluminescence reagents and images were captured using a Bio-Rad ChemiDoc XRS system. Far-Western blotting analysis Approximately 100 μg of each protein fraction was precipitated using ice-cold acetone, pelleted via centrifugation at 18,900

× g for 15 minutes, and air-dried at room temperature. The samples were then solublized by boiling in 1× SDS-PAGE sample buffer containing 2-mercaptoethanol. Duplicate 20 μL aliquots of each sample were buy AZD9291 subjected to 15% SDS-PAGE to separate the proteins based on their size. One set of the samples was then electrophoretically transferred to a PVDF membrane (Immobilon-Psq, Millipore). The PVDF membrane was pre-blocked with 1% BSA-TBST for 1 hour at room temperature to minimize non-specific protein binding and was then incubated in a solution of huPLG

(3 ug/mL in 1% BSA-TBST) for one hour with rocking at 37°C. Unbound PLG was removed by washing three times with TBST. Sheep anti-human PLG-specific antibody (diluted 1:2,000 in 1% BSA-TBST) was added (100 μL/well) and allowed to incubate for 1 hour at RT° with rocking. The PVDF membrane was washed three times with TBST to remove unbound primary antibody. The membrane was then incubated in a solution of anti-sheep/goat IgG monoclonal antibody (GT-34, diluted 1:5,000 in 1%BSA-TBST) with rocking for 1 hr at room temperature. The PVDF membranes were washed three times with TBST to remove unbound secondary antibody. The blot was developed using Pierce PicoWest chemiluminescence reagents and imaged using a Bio-Rad ChemiDoc XRS system. Proteomic identification of PLG-binding FT proteins Protein bands were excised from Coomassie-stained SDS-PAGE gels, cut into small pieces, incubated in 50% acetonitrile/100 mM ammonium bicarbonate until colorless, and dried via vacuum centrifugation.