Eight commonly used cell lines, including COS-7 (fibroblast-like kidney cells), NIH3T3 (mouse embryonic fibroblast cell line), HEK293 (human embryonic kidney cells), VERO (fibroblast-like kidney cell from African green monkey), HeLa (human epithelial carcinoma cell line), BHK (baby hamster kidney fibroblasts), HepG2 (human hepatocellular liver carcinoma cell line) and AGS (human gastric adenocarcinoma) were used in the study. All the cell lines were purchased from the National Cell Bank of the Iran Pasture Institute (Tehran, Iran). RPMI-1640 supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 10 µg/ml streptomycin (PAA Laboratories GmbH, Pasching, Austria) was utilized as the medium. The cells were cultured in humidified conditions at 37°C and in 5% CO₂.

Following the exponential phase of growth, the cells were washed twice by ice-cold phosphate buffered saline (PBS), and adherent cells were scraped off from the flask by a cell scraper. Following this, all cells were resuspended in 1 ml of radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), which included 2 mM phenylmethylsulfonyl fluoride, 10 µl of protease inhibitor cocktail and 1 mM sodium orthovanadate (Santa Cruz Biotechnology, Inc.). After centrifugation at 10,000 × g at 4°C for 20 min, cell debris was removed and the supernatant was used for western blotting. The Bradford assay protocol was utilized in order to determine the protein concentrations (16).

Equal amounts of total protein from each cell line were separated on a 12.5% discontinuous sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in a Mini-PROTEAN^® Tetra Handcast System (Bio-Rad, Hercules, CA, USA) for 90 min at 120 V. Subsequently, the separated proteins were transferred to a polyvinylidene difluoride membrane (Santa Cruz Biotechnology, Inc.) in a tank-transfer system (Bio-Rad) at 100 V for 60 min in the presence of 0.1% SDS. Following this, the membrane was blocked with 5% skimmed dry milk in Tris-buffered saline (pH 7.6) with 0.1% Tween-20 for 1 h at room temperature. The membrane was incubated using LGR5 mouse monoclonal antibody clone 2A2 (OriGene Technologies, Rockville, MD, USA; cat. no. TA503316), which was used as a specific primary antibody (diluted to 1:2,000).

The blots were washed three times in PBS-Tween and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Inc.; cat. no. SC-2005) secondary antibody was used for visualizing the antibody-antigen complex. The blots were developed with a Supersignal West Pico Chemiluminescent Substrate kit (Pierce, Rockford, IL, USA) for 5 min and images were captured by a Gbox device (Syngene, Cambridge, UK). To correct for protein loading and transfer efficiency, β-actin was used as the reference proteins for normalization in western blotting. By comparing with known protein size markers, the molecular weights were determined. Western blot band densities were quantitated by the GeneTools software (Syngene).

The expression pattern of the LGR5 levels in certain commonly used laboratory cell lines, which were AGS, HeLa, HEK293, HepG2, BHK, VERO, COS-7 and NIH3T3, was assessed. Western blotting on the total cell lysate was carried out to determine whether LGR5 was expressed at protein levels in these cells. All the cell lines tested showed a detectable amount of LGR5 expression; however, the level of expression differed in these cells. Expression of the LGR5 protein in the different cell lines in comparison with β-actin is shown in Fig. 1. A high level of LGR5 expression was detected in the AGS, BHK, VERO and NIH cell lines, while expression was barely detected in the other tested cell lines.

To estimate the relative expression of LGR5 in the tested cell lines, the band density for each cell line was determined using densitometry (Table I). The results of the normalized band densities showed that, as expected, the ASG cells expressed a higher level of LGR5 compared to the other tested cell lines. The BHK cells showed a higher level of LGR5 expression compared to the AGS cells. AGS cells are gastrointestinal cancer cells that are known to have an extremely high level of LGR5 expression, while BHK is an immortalized normal kidney cell of hamsters. Different amounts of LGR5 expression levels were also detected in two kidney-derived cells from monkey; VERO and COS-7 cells. VERO cells had a higher expression of LGR5 in comparison to COS-7 cells, which may reflect their distinct cell lineage in the kidney.