Chemicals and Reagents
Bovine serum albumin was obtained from Fisher Scientific (Loughborough, UK). Endoglycosidase H (Endo H) was purchased from New England Biolabs (Hertfordshire, UK). The antibodies and their sources were as follows: Antibodies for immunofluorescence: mouse anti-HA-Tag monoclonal antibody (dilution 1
200; Cell Signaling Technology), rabbit anti-calnexin polyclonal antibody (dilution 1
500; StressGen Biotechnologies), Alexa Fluor 568-goat anti-mouse IgG (dilution1
200; Molecular Probes), Alexa Fluor 488-goat anti-rabbit IgG(dilution 1
200; Molecular Probes). Secondary antibodies for Western blotting were as follows: rabbit-anti-goat Ig-horseradish peroxidase (Zymed Laboratories Inc., San Francisco, CA); sheep-anti-mouse Ig-horseradish peroxidase (Amersham Biosciences UK, Chalfont St Giles, UK).
Construction of the HA-tagged Endoglin construct
The HA-tagged version of the wild-type endoglin was generated by sequential site-directed mutagenesis to introduce the nine amino acids of the HA tag (YPYDVPDYA) in two cycles using pCMV5-ENG as a template (a kind gift of Professor Michelle Letarte, University of Toronto, Canada). In the first cycle five amino acids (YPYDV) of the HA tag were introduced prior to the stop codon of the Endoglin gene using the following mutagenesis primers.
In the second cycle, the above construct was used as a template and the remaining four amino acids (PDYA) of the HA tag were introduced using the following mutagenesis primers.
Generation of missense Endoglin mutants
The missense mutants were introduced by QuickChange site-directed mutagenesis with Turbo Pfu polymerase (Stratagene) and using the C-terminally HA-tagged Endoglin as templates. The primers for the site-directed mutagenesis to introduce the missense mutants are as follows with the mutagenic nucleotide in bold and underlined:
ENG-L32R- F: 5′GTCCATTGTGACCGTCAGCCTGTGGG3′
ENG-L32R- F: 5′CCCACAGGCTGACGGTCACAATGGAC3′
ENG-I271N-F: 5′ CACAACATGCAGAACTGGACCACTGGAG 3′
ENG-N423S-R: 5′GACCACCGCCTCACTGCTGATCATA 3′
All constructs were confirmed by direct DNA sequencing of the purified plasmids.
Sequencing was performed using the dideoxy method by fluorescent automated sequencing on the ABI 3130xl genetic analyzer (Applied Biosystems, Foster City, USA). The sequence data were analysed using Sequencing analysis software v5.3 (ABI, Foster City, USA).
Cell culture and transfection
HeLa cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin at 37°C with 10% CO2. For transfection, cells were grown on sterile cover slips in a 24 well tissue culture plates. Transfection was performed after 24 h using the liposomal transfection reagent FuGENE (HD Reagent, Promega, USA)according to manufacturer's instructions. For each well, 0.55 µg of the Endoglin wild type and the mutant constructs were used to transfect the cells. Twenty four hours after transfection, the cells were fixed and processed for microscopy.
Human embryonic kidney (HEK) 293 cells (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium/F12 medium (Invitrogen) supplemented with 10% fetal bovine serum(FBS) (Sigma Chemical Co, MO, USA), penicillin (10 units/ml) and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere of 5% CO incubator. One day before transfection, cells were seeded on six multiwells (Ø35 mm) and grown to 70% to 80% confluency. Transfection was performed, using FuGENE HD Transfection Reagent (Promega, USA) according to the manufacturer's instructions. Cells cultured were transfected with 2 µg of Endoglin wild-type or mutant plasmid DNAs.
For immunofluorescence, cover slip-grown HeLa cells were washed with phosphate-buffered saline (PBS), fixed by methanol at −20°C for 4 minutes. Fixed cells were then washed in PBS three times and incubated in blocking solution (1% BSA in PBS) for 30 min at room temperature. The fixed cells were then incubated for 1 h at room temperature with either mouse monoclonal anti-HA antibodies alone or co-incubated with rabbit polyclonal anti-calnexin antibodies. After washing with PBS, the cells were incubated with the appropriate fluorescently-labelled secondary antibodies for 1 h at room temperature then washed several times with PBS and mounted in immunofluor medium(ICN Biomedicals). We used EGFP-hRas as a marker for the plasma membrane. The H-Ras in this case was co-tansfected with the endoglin construct. Data were acquired using a Nikon confocal microscope 1(Japan). For presentation purposes, images were pseudo colored as either red (Endoglin) or green (calnexin and EGFP-H-Ras), contrast enhanced and overlayed using Adobe Photoshop® (Adobe Inc.). All images presented are single sections in the z-plane.
Protein extraction and Immunoprecipitation
Immuoprecipitation was used to isolate the expressed Endoglin from cell lysates of HEK293 cells. Forty eight hours post-transfection of cell transfection, culture media was removed and cells were gently washed twice with ice cold PBS (Phosphate-buffered saline). After washing, 150 µl of 1× ice-cold lysis buffer (Cell-Lytic Mcell Sigma C2978 lysis reagent) containing protease inhibitors (SIGMAFAST™ Protease Inhibitor Cocktail Tablets, EDTA-Free; Sigma S8830) was added to the 3.5 cmPetri dishes and incubated on ice for 5 min. Cells were then scraped off the plate and transferred to microcentrifuge tubes and kept on ice for 15 min.The cell lysates were then centrifuged at 14000 g for 15 min at 4°C.The supernatants were then transferred to a new tube without disturbing the pellet. Protein was quantified by Pierce Bicinchoninic Acid protein Assay kit according to the manufacturer's instructions. Ten microliters of protein A was added to 100 µl of cell lysate and incubated on a rotator for 45 min at 4°C. After centrifugation at 2500 g for 3 min the supernatant was transferred to a fresh 1.5 ml tube taking care not to transfer any beads slurry. Then 1 µl of anti-HA antibodies were added to the supernatants and incubated at 4°C on a rotator for 3 h then 10 µl of protein A (pre equilibrated in the corresponding IP lysis buffer) was added and incubated at 4°C for 2 h to capture the immune complex. The tubes were centrifuged at 2500 g for 30 s at 4°C and the supernatant was carefully removed and the beads were washed once with 100 µl of cold lysis buffer.
Endoglycosidase H Digestion
After immunoprecipitation, 10 µl of protein A-Sepharose beads containing the immune complex (Endoglin and anti HA antibody) was diluted by adding 10 µl of water. Then 5 µl of sodium phosphate buffer (250 mM, pH 5.5) and 1.25 µl of denaturation (containing 2%SDS and 1 M 2-mercaptpethanol, Sigma product number S4927) solution added to each sample. Samples were then denatured by heating at 100°C for 5 min. After cooling, 2 µl of Endoglycosidase H (Sigma, Saint Louis, USA) was added to each sample and incubated at 37°C for 3 h. The cleavage products were subjected to SDS-PAGE and western blotting using anti-HA antibodies as described below.
Western blotting analysis
Thirty µg of protein from control (untransfected cell), endoglin wild type and mutant constructions were subjected to15% SDS-polyacrylamide gel and transferred tonitro cellulose membrane. After 1 h blocking in 5% BSA (Bovine Serum Albumin), nitrocellulose blots were incubated overnight with anti-HA antibody (1
1000, Santa Cruz Biotechnology).Following washing with TRIS-buffered saline/Tween-20 (0.05%), membranes were incubated with goat anti-mouse IgGHRP (1
50000, Santa Cruz Biotechnology)for 1 h and developed according to standard procedures.