Chemicals and materials
All chemicals were purchased from Sigma Aldrich [St. Louis, MO, USA], unless otherwise noted. Materials were mainly purchased from Corning [Corning, NY, USA], Bio-Rad [Hercules, CA, USA], EMD Millipore [Billerica, MA, USA], and Eppendorf [Hauppauge, NY, USA], unless otherwise noted.
All cell lines were purchased from the American Type Culture Collection [ATCC, Manassas, VA, USA]. Human lung carcinoma A549 cells (ATCC CCL185), human hepatocellular carcinoma HepG2 cells (ATCC HB-8065), Burkitt’s Lymphoma Ramos cells (ATCC CRL-1596), and murine pre-osteoblast MC3T3-E1 Subclone 4 cells (ATCC CRL-2593) were thawed every month and routinely passaged twice per week into 75 cm2 flasks (Corning, Corning, NY, USA) to maintain them in a logarithmic growth phase at 37 °C in a humidified atmosphere with 5 % CO2 (NuAire, Plymouth, MN, USA). A549, HepG2, and Ramos were cultured in MEM medium supplemented with 10 % heat-inactivated FBS (Invitrogen, Carlsbad, CA, USA), 2 mM glutamine, 10 mM HEPES, and 1.5 g/L sodium bicarbonate. MC3T3-E1 cells were cultured in MEMalpha medium (Invitrogen) without ascorbic acid, supplemented with 10 % heat-inactivated FBS. At 85 % confluence, cells were harvested using 0.25 % Trypsin-EDTA solution and were sub-cultured or collected for subsequent experimental analysis. Since this study does not involve humans, human data or animals, an ethics committee approval was not required.
Native western blot analysis
Cells were lysed with ProteoJET Mammalian Cell Lysis Reagent (Fermentas, Hanover, MD, USA), following the manufacturer’s protocol, to obtain total native conformation proteins. Protein concentrations were determined using a Bio-Rad Protein Assay Kit. A molecular weight (kDa) standard (NativeMark - Novex, Life Technologies, Grand Island, NY, USA) and equal amounts of protein extracts (50 μg) were subjected to electrophoresis on a 8 % SDS-free polyacrylamide gel (PAGE) and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane following the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). After blocking non-specific binding sites with 5 % skimmed milk, blots were incubated with primary rabbit polyclonal antibody specific to eIF3f (Biolegend, San Diego, CA, USA) or primary goat antibody specific to alpha 1B-ADR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and horseradish peroxidase-conjugated goat anti-rabbit (Biolegend) or rabbit anti-goat secondary antibody (Santa Cruz Biotechnology). The bound antibody was detected by enhanced chemiluminescence (ECL) on an X-ray film (GE Healthcare Life Sciences, Piscataway, NJ, USA).
Complex member identification
After native electrophoresis, the 120 kDa region of the gel was excised and eluted overnight at 4 °C in 1 mL of ProteoJet Mammalian Cell Lysis Reagent. For non-specific bound protein removal, the elution was incubated 1 h at 4 °C with a non-specific IgG and 100 μL of 50 % Protein A-Sepharose 4B beads (Invitrogen). After centrifugation, the supernatant was incubated at 4 °C with the antibody against eIF3f and 100 μL of 50 % Protein A-Sepharose 4B beads, washed 4 times with lysis buffer, resuspended in sample buffer (2 % SDS, 20 % glycerol, and 0.5 % bromophenol blue in 62 mM Tris HCl buffer, pH 6.8), boiled for 5 min, and subjected to electrophoresis on a 10 % SDS-polyacrylamide gel (10 % SDS-PAGE). A PageRuler Plus Prestained Protein Ladder (Thermo Scientific, Rockford, IL, USA) was included in all SDS-PAGEs. The resolved proteins were detected by Coomassie blue R250 staining (Bio-Rad); the unknown protein bands were excised and sent for N-terminal protein/peptide sequencing (Iowa State University of Science and Technology, Ames, IA, USA). After sequencing service, the partial amino acid sequences were subjected to a NCBI Blastp search to identify possible protein partners of eIF3f.
Immunoprecipitate western blot analyses
As describe above, after a native electrophoresis, the 120 kDa region of the gel was excised, eluted, cleared with a non-specific IgG and Protein A-Sepharose 4B beads and centrifuged. The supernatant was incubated with the corresponding primary antibody (anti-eIF3f or anti-alpha 1B-ADR) and Protein A-Sepharose 4B beads, washed, resuspended in sample buffer, boiled, and subjected to electrophoresis (10 % SDS-PAGE). Proteins from the gel were electrophoretically transferred to a PVDF membrane and blotted with anti-eIF3f or anti-alpha 1B-ADR antibodies, and the corresponding secondary horseradish peroxidase-conjugated antibodies. The bound antibody was detected by ECL on an X-ray film.
To express human eIF3f, we used the previously described pSK11F plasmid . To express alpha 1B-ADR, we used plasmid AR0A1B0000 (Missouri S&T cDNA Resource Center, Rolla, MO, USA). To obtain plasmid eIF3fΔ91 (eIF3f lacking first 91 AA of the N-terminal region), template pSK11F and the forward 5’-CCCTTCCCCGGCGGCAGCATGGTC-3’ and reverse 5’-CAGGTTTACAAGTTTTTCATTG-3’ oligonucleotides were used to amplify the eIF3f coding sequence corresponding to amino acids 92–357. The forward oligo was designed to contain a classic Kozak consensus sequence (see underlined nucleotides), by modifying only 2 nucleotides. The amplicon was cloned in pGEM vector (Promega, Fitchburg, WI, USA) and verified by DNA sequencing (Elim Biopharmaceuticals, Hayward, CA, USA).
[gamma-32P]GTP Binding Assay
Membranes from A549 human cells were obtained using ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem, La Jolla, CA, USA), as described by the manufacturer. To obtain membranes with over expressed alpha 1B-ADR, before membrane extraction, cells were transiently transfected (48 h) with plasmid AR0A1B0000 using LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s specifications and as described previously . For in vitro translation of eIF3f and eIF3fΔ91, mRNA was synthesized in vitro using T3 and T7 RNA polymerase (Invitrogen), respectively, and according to the manufacturer’s instructions. The respective proteins were synthesized in vitro with a Rabbit Reticulocyte Lysate System (Promega), using 1 μg of mRNA. For the [gamma-32P]GTP binding assay , 20 μg of membrane protein and 5 μL of the translation reaction were resuspended in 55 μL of 50 mM Tris–HCl (pH 7.4), 2 mM EDTA, 100 mM NaCl, 1 μM GDP, 3 mM MgCl and 30 nM the [gamma-32P]GTP (Institute of Isotopes Co Ltd, Budapest, Hungary). As a control, the reaction was also performed using water instead of a mRNA. The reactions were incubated at 30 °C for 5 min in the presence or absence of agonist (100 nM adrenaline). The reaction was terminated by adding 600 μL of ice-cold stop solution (50 mM Tris–HCl pH 7.5, 20 mM MgCl2, 150 mM NaCl, 0.5 % Nonidet, 100 μM GDP, and 100 μM GTP) and incubating for 30 min in ice. To each reaction, non-specific IgG and 100 μl of Protein A-Sepharose 4B beads were added and further incubated on ice for 20 min. Non-specifically bound protein was removed by centrifugation. The supernatant was then incubated 1 h at 4 °C with 1 μg of Gαq/11 antibody (Santa Cruz Biotechnology) and immunoprecipitated with 100 μl of Protein A-Sepharose 4B beads for 1 h at 4 °C. Immunoprecipitates were collected, washed 4 times in buffer without detergent, and resuspended in a TE buffer. The samples were analyzed in a scintillation counter (Wallac, Oy, Turku, Finland).
All experiments were independently repeated at least three times. Results of multiple experiments are expressed as mean ± standard error (S.E.). Analysis of Student’s t test was used to assess the differences between means. A p < 0.05 was accepted as statistically significant.