Supersignal west pico chemiluminescence system

Membranes were probed with primary antibodies for phosphorylated ERK (Cell Signaling Technology; 4370), total ERK (Santa Cruz Biotechnology; sc-153 or Cell Signaling Technology; 4696), sheep polyclonal anti-DUSP5 (13 (link)), rabbit anti-DUSP5 (Abcam; ab200708), c-Myc (Santa Cruz Biotechnology; sc-40), β-tubulin (Sigma; T8328), or α-tubulin (Santa Cruz Biotechnology; sc-23948 HRP). Horseradish peroxidase (HRP)-conjugated secondary antibodies (Southern Biotech) were used at a concentration of 1:2,000. SuperSignal West Pico chemiluminescence system (Thermo Scientific) and a FluorChem HD2 Imager (Alpha Innotech) were used to detect proteins. For indirect immunofluorescence, anti-DUSP5 antibody (Abcam) was diluted 1:100 in phosphate-buffered saline with 0.1% Tween-20 (PBS-T) containing 1% BSA and incubated overnight. Secondary goat anti-rabbit IgG conjugated to Alexa Fluor 594 (Thermo Fisher; A-11012) and DAPI were used for detection of DUSP5 and nuclei, respectively.

After experimental treatments and incubation periods, NRVMs were washed with ice cold PBS and lysed with PBS containing 300 mM NaCl, 0.5% Triton-X and HALT™ protease/phosphatase inhibitors. NRVM lysates were sonicated and centrifuged to then separate the supernatant and pellet. The supernatant was isolated and used for immunoblotting experiments. BCA reagents were used to measure protein concentration of all samples as to normalize to 10 μg per sample for SDS-PAGE. Samples were resolved with SDS-PAGE and transferred onto a nitrocellulose membrane which was then incubated overnight in a primary antibody cocktail of BSA (2.5%) in 1x Tris-Buffered Saline and Tween^® (TBST). The next day, membranes were washed in TBST prior to being exposed to horseradish peroxidase-conjugated secondary antibodies (Southern Biotech, Birmingham, AL, USA). Finally, SuperSignal West Pico Chemiluminescence System (Thermo Fisher Scientific) was used on membranes prior to exposure on a ChemiDoc XRS+ Imager (BioRad, Hercules, CA, USA).

HT-1080 cells in 6-well plates at 70–80% confluence were incubated overnight in the presence or absence of A. muricata aqueous extract 100 µg/mL. Then, conditioned media was collected, and cells were lysed in 100 µL of RIPA lysis buffer (Sigma/Merck, Darmstadt, Germany). The protein concentration of the samples was estimated using a Bradford assay [38 (link)], and a volume corresponding to 30 μg of total protein of cell lysates, and the equivalent volume of conditioned media, were subjected to SDS-PAGE denaturing electrophoresis.
After electrophoresis, gels were electrotransferred to a nitrocellulose membrane. Membranes were blocked in TBS-T buffer (20 mM Tris, 137 mM NaCl, 0.1% Tween-20) containing 5% semi-skimmed milk and then incubated overnight with rabbit monoclonal anti-transferrin receptor (Cell Signaling Technology, Danvers, MA, USA) antibody diluted 1:500–1000 in TBS-T with 5% BSA. After incubation with the secondary antibody diluted 1:5000 in blocking buffer, the signal was detected using the SuperSignal West Picochemiluminescence system (Thermo Fisher Scientific, Waltham, MA, USA) and an imaging system Chemidoc XRS (Bio-Rad, Hercules, CA, USA). The same membranes were incubated with the anti-tubulin antibody at a dilution of 1:1000. Blots were quantified by densitometry with the software FIJI.