cDNAs and constructs
A human cDNA clone [Genbank AB037794] was used as described [24, 25]. To generate wild type AMSH-2 expression vector with a carboxy-terminal Myc epitope, we subcloned AMSH-2 open reading frame into the Nco I and Xho I sites of pEF/myc/cyto (Invitrogen, Carsbad, CA) by standard PCR procedures using the primers: AAAACCATGGATCAGCCTTTTACTGTGAATTC (5' primer) and AAACTCGAGCTGTTCAGATGGTGATGATGAC (3' primer). To generate AMSH-2 V5 carboxy-terminal-tagged expression vector, the open reading frame of AMSH-2 was subcloned into Nco I and Xho I sites of pEntr4 (Invitrogen) using the same 5' primer as described above and AAACTCGAGAGCTGTTCAGATGGTGATGATGACCTAG (3' primer), and transferred to pEF1/V5-HisB (Invitrogen), which has been previously made gateway compatible by inserting a cassette in the EcoRV site.
3TP-lux, PAI-1-luc, pELAMP-luc+, TRAF2, Flag-tagged Smad7, Smad3, have been previously described [6, 20, 36–38, 45] while Flag-tagged Smad2 and HA-tagged Smad4 were generously provided by Joan Massague.
Northern blot analysis
Human tissue northern blot II and cancer cell line blot were obtained from CLONTECH (Palo Alto, CA). We isolated and radiolabeled an AMSH-2 cDNA (nucleotides 1088–2010) produced by digestion of AMSH-2 cDNA clone with HindIII and SphI. Hybridization signals were detected on a BAS-2000 bioimaging analyzer (Fuji film, Tokyo, Japan). After exposure, the blots were stripped and reprobed with beta-actin to control for equal amounts of PolyA+ RNA loading.
Cell culture and antibodies
293 and 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and penicillin/streptomycin. HepG2 cells were grown in MEM supplemented with 10% fetal bovine serum, non essential amino-acids, sodium pyruvate and penicillin/streptomycin.
The peptide corresponding to the C-terminus of human AMSH-2 was synthesized by Boston Biomolecules, Inc (Woburn, MA). The specific AMSH-2 rabbit polyclonal antibody (anti-AMSH-2) was raised at Covance Research Products Inc. (Denver, PA). The following antibodies were obtained: (i) c-Myc mAb 9E10 (Covance Research Products Inc);(ii) anti-V5 and anti-V5 HRP antibodies (Invitrogen); (iii) anti-Flag HRP (UPSTATE, Lake Placid, NY);and (v) anti-HA 12CA5, anti-HA HRP (Roche Diagnostics Corp, Indianapolis, IN).
Metabolic labeling and immunoprecipitation
To test endogenous expression of AMSH-2, 293T and HepG2 cells were metabolically labeled with 35S methionine and 35S cysteine and lysed in modified RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate) with 1 mM sodium orthovanadate and protease inhibitors. Samples were incubated either with pre-immune serum or AMSH-2 antiserum. Immunoprecipitated proteins were resolved by SDS-PAGE. To test expression of the Myc-tagged AMSH-2 construct, cells were transfected with 15 μg of DNA using the standard calcium phosphate method. Twenty-four h postransfection, the cells were metabolically labeled for 16 h. and subsequently lysed and immunoprecipitated with anti-Myc antibody as above.
HepG2 cells were transfected with 0.5 μg of the corresponding luciferase reporter, 50 ng of β-galactosidase reporter and 4.5 μg of DNA. Twenty-four h postransfection, the cells were depleted from serum and stimulated for 16–20 h with 5 ng/ml of purified recombinant human TGF-β1 (R&Dsystems. Inc. Minneapolis, MN). Luciferase and β-galatosidase activities were measured according to manufacturer's instructions (Tropix, Bedford, MA). Measurements were corrected by β-galactosidase activity. The experiments were repeated at least three times.
293T cells were cotransfected with 13 μg of AMSH-2 V5 construct and 2 μg of the corresponding Smad constructs. After 48 h, the cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1 mM EDTA, 1% NP-40) with 1 mM sodium orthovanadate and protease inhibitors. Lysates were incubated with anti-V5 antibody, and the resulting immunocomplexes were separated in SDS-PAGE. Blots were probed with the indicated HRP conjugated antibody.
To target AMSH-2 and Smad 7, we designed 21 nt siRNA duplexes: AACAATTCCTTGCTGAATGTA and AACCGCAGCAGTTACCCCATC, according to . All siRNA duplexes including lamin A/C siRNA duplexes and scrambled siRNA were obtained from Dharmacon Research. Inc. (Lafayette, Colorado).
HepG2 cells grown in 6 well plates were transfected with 250 ng of 3TP-lux reporter, 50 ng of β-galactosidase reporter and 0.12 nmol of the corresponding siRNA duplex. After 48 h of transfection cells were depleted from serum and treated with 5 ng/ml TGF-β1. Luciferase and β-galactosidase activities were measured after 17 h stimulation.