For the assessment of enzyme
levels, 40 μg of cellular proteins were resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot
analysis of TS and DHFR was conducted as previously described,61 (link) using a 1:250 dilution of the anti-human TS
mouse TS106 monoclonal primary antibody (Abnova, Italy), and 1:250
dilutions of the anti-human DHFR mouse A-4 monoclonal antibody (Santa
Cruz Biotechnology, Inc.). Cells were plated in complete medium containing
10% heat-inactivated FBS and after 24 h, in FF medium (pH 7.2), except
for the control sample (CTRL). After an additional 24 h, the cells
were treated with the FA–peptide conjugates or PMX. For the
assessment of FRα levels, nonreducing and nondenaturating conditions
(no SDS) were used. Gels were blotted onto poly(vinylidene difluoride)
(PVDF) membranes (Hybond-P, Amersham). Antibody staining was performed
with a chemiluminescence detection system (ECL Plus, Amersham), using
a 1:500 dilution of the anti-human mouse Mov18 monoclonal primary
antibody (Enzo Life Sciences) and 1:2000 dilution of anti-human β-actin
mouse AC-15 antibody (Santa Cruz Biotechnology, Inc.) in TBS-T with
5% dry milk for normalization, in conjunction with a 1:1500 dilution
of a horseradish peroxidase-conjugated sheep anti-mouse secondary
antibody (Amersham).
levels, 40 μg of cellular proteins were resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot
analysis of TS and DHFR was conducted as previously described,61 (link) using a 1:250 dilution of the anti-human TS
mouse TS106 monoclonal primary antibody (Abnova, Italy), and 1:250
dilutions of the anti-human DHFR mouse A-4 monoclonal antibody (Santa
Cruz Biotechnology, Inc.). Cells were plated in complete medium containing
10% heat-inactivated FBS and after 24 h, in FF medium (pH 7.2), except
for the control sample (CTRL). After an additional 24 h, the cells
were treated with the FA–peptide conjugates or PMX. For the
assessment of FRα levels, nonreducing and nondenaturating conditions
(no SDS) were used. Gels were blotted onto poly(vinylidene difluoride)
(PVDF) membranes (Hybond-P, Amersham). Antibody staining was performed
with a chemiluminescence detection system (ECL Plus, Amersham), using
a 1:500 dilution of the anti-human mouse Mov18 monoclonal primary
antibody (Enzo Life Sciences) and 1:2000 dilution of anti-human β-actin
mouse AC-15 antibody (Santa Cruz Biotechnology, Inc.) in TBS-T with
5% dry milk for normalization, in conjunction with a 1:1500 dilution
of a horseradish peroxidase-conjugated sheep anti-mouse secondary
antibody (Amersham).