Chemiluminescent reagent

RIPA (Radio-Immunoprecipitation Assay) lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract proteins from cells, and protein concentration was established by Bradford method (Sigma–Aldrich, Madrid, Spain). Separation of proteins was carried out in 8% SDS-PAGE gels in which 40 µg of proteins of each sample were loaded previously heated at 65 °C for 20 minutes. Then, proteins were electrotransferred to a nitrocellulose membrane (45 µm pore size) (Millipore, Burlington, MA, USA) and blocked for 1 hour in 5% milk in TBS with 0.1% Tween-20 (TBS-T) (Bio-Rad, Hercules, CA, USA). After several washes in TBS-T, the incubation of the anti-LGR5 primary antibody (1:750) (Anti-LGR5 (OTI2A2): MA5-25,644; Invitrogen, Waltham, Massachusetts, USA) was performed overnight at 4 °C. Then, a secondary antibody (m-IgGκBP-HRP: sc-516,102; Santa Cruz Biotechnology, Dallas, Texas, EEUU) was added (1:2000) for 1 hour at room temperature. Chemiluminescent reagents (Amersham Biosciences, Saint Louis, MO, USA) were used for the detection of membrane-bound antibodies. Besides, anti-β-actin IgG (A3854, Sigma Aldrich, Madrid, Spain) (1:25,000 dilution) was used as an endogenous control. The open-source Fiji image analysis software was used to quantify the bands obtained to calculate relative protein expression.28 (link)

Western blotting was performed as described previously 4 (link). Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gels, transferred to poly-vinylidene difluoride membranes (Millipore, Billerica, MA), and finally immunoblotted with primary antibodies. Primer antibodies were used at 1:500 to 1:1000 dilution. Horseradish peroxidase-conjugated anti-mouse and rabbit secondary antibodies (Santa Cruz Biotechnology, Dallas, TX) were performed at 1:5000 dilution. Protein signals were detected by chemiluminescent reagents (Amersham Pharmacia, Piscataway, NJ).

Western blot analysis was performed as described previously (12 (link)). Primary antibodies against intercellular adhesion molecule-1 (ICAM-1; sc-1511), vascular cell adhesion molecule-1 (VCAM-1; sc-1504) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), fractalkine (TP-213; Torrey Pines BioLabs, Houston, TX, USA), phosphorylated (p)-p65 (#3033) and p65 (#4764; both from Cell Signaling Technology) were used. The membranes were reblotted with anti-actin antibody to verify the equal loading of protein in each lane. All signals were visualized using chemiluminescent reagents (Amersham Pharmacia Biotech, London, UK) and analyzed using a densitometric scanner (LAS-3000; FujiFilm, Tokyo, Japan).

Samples of equal amount of proteins were separated with 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in TBST buffer (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated with primary and secondary antibodies at room temperatures. The indicated protein bands were detected using chemiluminescent reagents (Amersham, Franklin Lakes, NJ, USA) (22) (link).