HeLa cells (1.5 × 106) were treated
with CNC and GNC (0, 200, 300, 400, and 500 μg/mL)
for 48 h. Proteins were extracted from the cells in the lysis buffer
(20 mM Tris–HCl pH 7.4, 2 mM EDTA, 150 mM NaCl, 1.2% sodium
deoxycholate, 1.2% TritonX-100, protease inhibitor cocktail (PIC))
and quantified by the Bradford method. An equivalent amount of proteins
(75 μg) were resolved by 15% SDS-PAGE and transferred onto a
nitrocellulose membrane (GVS Life Science) with a wet blot transfer
system (VWR). The membranes were blocked with 2–5% nonfat milk
in TBS-Tween 20 (0.2%) and incubated with primary antibodies (seeTable S1 for reagent details) with the following
dilutions: anti-Caspase 9 (1:1000) and anti-α-tubulin (1:5000).
Lysates (50 μg) of HFs-hTERT cells and their transformed variant
were resolved by 10% SDS-PAGE, processed as described above, and anti-H-Ras
(1:1000) and anti-SV40 LT (1:750) antibodies were used to detect the
corresponding proteins. Then membranes were washed with TBS-Tween
20 (0.2%) and incubated with the appropriate HRP-conjugated secondary
antibodies (1:10,000 dilutions). Immunoblots were developed using
the WesternBright chemiluminescent substrate (Advansta). Images were
recorded with a LI-COR Odyssey image recorder.
with CNC and GNC (0, 200, 300, 400, and 500 μg/mL)
for 48 h. Proteins were extracted from the cells in the lysis buffer
(20 mM Tris–HCl pH 7.4, 2 mM EDTA, 150 mM NaCl, 1.2% sodium
deoxycholate, 1.2% TritonX-100, protease inhibitor cocktail (PIC))
and quantified by the Bradford method. An equivalent amount of proteins
(75 μg) were resolved by 15% SDS-PAGE and transferred onto a
nitrocellulose membrane (GVS Life Science) with a wet blot transfer
system (VWR). The membranes were blocked with 2–5% nonfat milk
in TBS-Tween 20 (0.2%) and incubated with primary antibodies (see
dilutions: anti-Caspase 9 (1:1000) and anti-α-tubulin (1:5000).
Lysates (50 μg) of HFs-hTERT cells and their transformed variant
were resolved by 10% SDS-PAGE, processed as described above, and anti-H-Ras
(1:1000) and anti-SV40 LT (1:750) antibodies were used to detect the
corresponding proteins. Then membranes were washed with TBS-Tween
20 (0.2%) and incubated with the appropriate HRP-conjugated secondary
antibodies (1:10,000 dilutions). Immunoblots were developed using
the WesternBright chemiluminescent substrate (Advansta). Images were
recorded with a LI-COR Odyssey image recorder.