Fluorchem sp digital imager

Cell lysates from different cell lines were collected in RIPA buffer, which contained a protease inhibitor cocktail (Santa Cruz Biotechnology, Inc). The BCA assay was used to determine protein concentrations. Samples of 40 µg of protein were separated using SDS‐PAGE and then transferred to nitrocellulose membranes that were then blocked in 5% (w/v) nonfat dry milk in Tris‐buffered saline‐Tween 20 (TBST). After 2 hours of blocking, the membranes were incubated with a rabbit PAK‐1 antibody (Cell Signaling Technology) at a dilution of 1:1000 in 1% (w/v) BSA TBST overnight. The antibody against GAPDH (Santa Cruz Biotechnology Inc) was used at a dilution of 1:4000 in 1% (w/v) BSA in TBST for 1 hour. Membranes were then incubated with the appropriate peroxidase‐conjugated secondary antibodies (Promega,) used at a dilution of 1:2500. The membranes were then washed with TBST three times for 10 minutes each. Bands were visualized using chemiluminescent substrates (Thermo Scientific) and imaged with a FluorChem SP digital Imager (Alpha Innotech). Immunoblot analysis was performed on protein samples from at least three different passages (n = 3) of control and KD cells. Densitometry was performed using National Institutes of Health Image J software.

Cell lysates from different cell lines were collected in RIPA buffer, which contained a protease inhibitor cocktail (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The concentration of proteins in different samples was determined using the BCA assay. Cell lysates were first separated using gel electrophoresis and then transferred to nitrocellulose membranes and blocked for 2 h. The nitrocellulose membranes were incubated with a rabbit sPLA₂ IIA antibody (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:500 in TBS-T with 1% (w/v) BSA overnight. The antibody against GAPDH (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was used at a dilution of 1:4000 in 1% (w/v) BSA in TBS-T for 1 h. Membranes were then incubated with the appropriate peroxidase-conjugated secondary antibody (Promega, Madison, WI, USA) used at a dilution of 1:2500. Membranes were then washed with TBS-T three times for 10 min. Bands were developed using chemiluminescent substrates for horseradish peroxidase (Thermo Scientific, Waltham, MA, USA) and visualized using a Fluorchem SP digital imager (Alpha Innotech, San Leandro, CA, USA). Densitometry to quantify immunoblot bands was performed using the National Institutes of Health Image J software (ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA).