Supplementary Materials and methods.
Measurement of TGF-β1
Cells were seeded in 24-well plates at 5105 cells/well. After 48 h incubation, cells were
washed with PBS and then incubated with serum-free medium for 24 h. Cell-free supernant
was collected and he amounts of TGF-β1 in the culture supernatant were measured by
immunoassay kits according to the manufacturer’s protocol (KOMABIOTECH, Seoul,
Korea).
Reverse transcription-PCR (RT-PCR) assays
We used the following primers to measure of TβRI and TβRII RNA levels: TβRI, 5′GGTCTTGCCCATCTTCACAT-3′ (sense) and 5′-TCTGTGGCTGAATCATGT CT-3′
(antisense);
TβRII,
5′-GTCTACTCCATGGCTCTGGT-3′
(sense)
and
5′-ATCTGG
ATGCCCTGGTGGTT-3′. GAPDH was also amplified as the reference gene using the
following primers: GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ (sense) and 5′TCCACCACCCTGTTGCTGTA-3′ (antisense).
Reporter assays
Briefly, cells were transfected with the indicated reporter plasmid in combination with the
indicated constructs. The cells were treated with or without TGF-β1 for an additional 24 h
after transfection. The luciferase assay was performed using the Dual-luciferase reporter
assay system according to the manufacturer’s instructions (Promega, Madison, WI, USA).
Results were normalized to the Renilla activity expressed by the cotransfected Rluc gene
under the control of a constitutive promoter.
Small hairpin RNA
Lentiviral vector containing validated small hairpin RNA (shRNA) for syntenin (Cat #
TL309594) as well as the control shRNA vector with non-effective 29-mer scrambled
sequence were purchased from Origene Technologies, Inc. (Rockville, MD, USA). Cells
were infected with lentiviral shRNA as recommended by the manufacturer and after
puromycin selection, stable selected cells were used for the experiments. Efficiency of
syntenin knockdown was determined in resistant cells by Western blot analyses.
Cell surface protein biotinylation
Cell surface protein biotinylation assay was performed using water-soluble membrane
impermeable EZ-Link sulfo-NHS-LC-biotin reagent, according to the manufacturer’s
instructions (Pierce, Rockford, IL, USA). Cells were washed with ice-cold PBS and
incubated with EZ-link Sulfo-NHS-LC-Biotin (Pierce, 0.5 mg/ml in PBS). Biotinylation was
stopped with 0.1 M glycine in PBS, and cells were lysed in lysis buffer (50 mM Tris-HCl, pH
7.4, 150 mM NaCl, 1 mM EDTA, 5 mM sodium orthovanadate, 1% NP-40 and protease
inhibitors cocktail). The biotinylated protein was pulled down by incubating with neutravidin
beads at (Pierce) at 4°C overnight and analyzed by immunoblotting using anti-TβRI antibody
(Santa Cruz) or transferrin (TfR) antibody.