Ecl pico plus chemiluminescent reagent

Ten micrograms of KM12C, KM12SM, KM12L4a, SW480, and SW620 protein extracts was separated on 10% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes (100 V, 90 min). The membranes were blocked for 1 h using 3% BSA in 0.1% Tween-PBS, and alternatively incubated overnight at 4 °C with the biotinylated goat anti-human Hp antibody (Btn-Ab, 1/1000, component of the DuoSet®, DY8465-05, R&D Systems) or anti-RhoGDi antibody (1/1000; Santa Cruz Biotechnology) in the same solution. After three washes with 0.1% Tween PBS (PBST), the membranes were incubated with either 1/500 Strep-HRP (RayBiotech) or 1/1000 HRP-anti mouse IgG (Sigma), respectively, for 1 h at room temperature. The membranes were then washed thrice with PBST, and a luminescence signal was developed with the ECL Pico Plus chemiluminescent reagent (Thermo Fisher Scientific) and detected on an Amersham Imager 680 (GE Healthcare).
The intensity of each WB lane was qualitatively measured using ImageJ. Hp and RhoGDi protein bands were normalized using the loading control (RhoGDi).

For western blot (WB) analysis, 10–15 µg of each protein extract were separated on 10% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes at 100 V during 90 min. 40–60 µg of secretome proteins in 100 µL PBS were dot blotted onto nitrocellulose membranes using the Bio-Dot 96-Well Microfiltration (Bio-Rad).
Then, membranes were blocked with 0.1% Tween PBS 1x containing 3% skimmed milk (blocking buffer) during 1 h at RT and incubated with primary antibodies at optimized dilutions (Supplementary Table 4) in blocking buffer O/N at 4ºC. Then, membranes were washed three times with 0.1% Tween PBS 1x and incubated with the appropriate indicated HRP-conjugated secondary antibodies (Supplementary Table 4) diluted in blocking buffer during 1 h at RT. Next, membranes were washed three times with 0.1% Tween PBS, and, finally, signal was developed using the ECL Pico Plus chemiluminescent reagent (Thermo Fisher Scientific) and detected on an Amersham Imager 680 (GE Healthcare). Protein band intensities were quantified using ImageJ Software.