Cell lines and Reagents
Colon cancer cell lines LoVo, SW480 and HCT116 were obtained from ATCC (Manassas, VA) and maintained in DMEM (Invitrogen, Carlsbad, CA). Gastric cancer cell BGC823 was maintained in RPMI-1640 medium (Invitrogen). The medium was supplemented with 10% fetal calf serum (Invitrogen). The antibodies against integrin β1 (MAB 2000), integrin α1 (MAB1973) and phosphorylated tyrosine (4G10) (16–316) were from Millipore (Billerica, MA). Anti-phosphorylated integrin β1 (Tyr783) (600601) and (Tyr795) (600501) antibodies were obtained from Biolegend (San Diego, CA). Antibody against PRL-3 (clone 318) was from Santa Cruz (Santa Cruz, CA). GADPH antibody was from Proteintech Group (Chicago, IL). PRL-3 inhibitor 1-(2-bromobenzyloxy)-4-bromo-2-benzylidene rhodanine (P0108) was from Sigma (St. Louis, MO). The tyrosine phosphatase inhibitor (Pervanadate) was freshly prepared by dissolving sodium orthovanadate (Sigma) with PBS to 30 mM, adding hydrogen peroxide at 0.18% (v/v), and incubating for 15 min at room temperature avoiding light before treating the cells at the final concentration of 100 μM.
Plasmids transfection and RNA interference
The plasmids expressing myc-PRL-3, myc-PRL-3-mt (cystine 104 was mutated to serine) and GFP-PRL-3 were described as previously
]. The small interference RNAs (siRNAs) targeting integrin α1 and PRL-3 were synthesized by GenePharma (Shanghai, China), the sequence for integrin α1: sense, 5’- GCCCUUAUAUGCCUAUAGA -3’; antisense, 5’- UCUAUAGGCAUAUAAGGGC -3’; the sequence for PRL-3: sense, 5’- CAGCAAGCAGCUCACCUAC -3’; antisense, 5’- GUAGGUGAGCUGCUUGCUG -3’. Plasmids and siRNA were transfected into cells with Lipofectamine 2000 (Invitrogen) following provider’s instruction.
To visualize the localization of integrin β1, BGC823 cells were cultured on the coverslips and fixed with 2% paraformaldehyde for 30 min at 4°C, followed by permeabilization with 0.5% Triton X-100 in PBS for 5 min, and blocking with 3% bovine serum albumin overnight at 4°C. After incubation with anti-integrin β1 (1: 300) for 1 hr at room temperature, cells were probed with tetramethyl rhodamine isothiocyanate-conjugated secondary antibody, counterstained with 4', 6-diamidino-2-phenylindole (DAPI), and mounted on 50% glycerol/PBS. Localization of PRL-3 was analyzed by transfecting the cells with pEGFP-PRL-3. A Leica SP2 confocal system (Leica Microsystems, Dresden, Germany) was used to observe the localization of integrin β1 and GFP-PRL-3.
Western blotting and immunoprecipitation
For Western blotting, cells were directly lysed in 1x loading buffer. For immunoprecipitation assay, cells were homogenized in lysis buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 50 mM NaF, 1 mM Na3VO4, 2 mM dithiothreitol, 1 × protease cocktail (Sigma)) for 20 min at 4°C. The supernatant was collected after centrifugation at 12,000 × g for 20 min at 4°C and then incubated with indicated antibodies conjugated to protein G-Sepharose (Invitrogen). Cell lysates or immunoprecipitates were separated by SDS-PAGE and electro-blotted to the nitrocellulose membranes. Non-specific binding was blocked with 5% non-fat milk in PBS overnight at 4°C and was rinsed twice with PBST. Then the membranes were incubated with indicated primary antibodies at room temperature for 1.5 h, and washed six times with PBST, followed by horseradish peroxidase-labeled secondary antibodies for 45 min and washed again as above. Protein bands were visualized with enhanced chemoluminescence system (Thermo Scientific, Rockford, IL).
GST Pull-down assay
Deoxyribonucleic acids encoding intracellular domain of integrin β1 (752-798aa) was amplified by PCR with the following primers: sense, 5’- GACTGAATTCAAGCTTTTAATGATAATTCATG -3’; antisense, 5’- AGCAACTCGAGGTGTTGTGGGATTTGCAC -3’. The PCR product was digested by EcoR I/Xho I and inserted into pGEX-4T1 vector. His-tagged PRL-3 was constructed by inserting the digested PRL-3 into pET-28a vector. For pull-down assay, glutathione-Sepharose beads (Invitrogen) were incubated with E. coli bacteria lysates expressing GST-integrin β1 or GST. After being washed with PBS, these beads were incubated with His-PRL-3 in binding buffer (10 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 mM dithiothreitol, 1 mM PMSF, 1% Triton X-100, 10% glycerol) for 4 hours at 4°C, then washed three times, boiled in SDS loading buffer, separated by SDS-PAGE and immunoblotted with PRL-3 and GST antibodies.
In vitro phosphatase assay
Integrin β1 protein was immunoprecipitated from 1000 μg protein from HCT116 cell lysates with 1 μg integrin β1 antibody conjugated to protein G-Sepharose. For control, cell lysates were immunoprecipitated with 1 μg pre-immune IgG. After being washed 3 times with lysis buffer, once with dephosphorylation buffer (50 mM Tris pH 7.5, 2 mM dithiothreitol, 1 mM MgCl2, 0.1 mM MnCl2), the precipitates were used as substrate for in vitro phosphatase assay. GST-PRL-3, GST-PRL-3-mt, and GST (1 μg each) were used as phosphatase and incubated with substrate in 20 μL dephosphorylation buffer at 30°C for 30 min. The reaction was stopped by boiling in 2 × loading buffer and the phosphorylated integrin β1 was detected by immunoblotting with 4G10 antibody.