Cells were seeded at a density of 2 ×
105 in 6-well plates, allowed to adhere overnight, before
incubation with DMSO, 10 mM BCH, 50 μM ESK242, or 50 μM
ESK246 for 6 h or 3 d. Cells were lysed by the addition of lysis buffer
(200 μL) with protease inhibitor Cocktail III (Bioprocessing
Biochemical) and 1 mM Na3VO4 (Sigma). Equal
protein (micro-BCA method; Pierce, IL) was loaded on 4–12%
gradient gels (Life Technologies), electrophoresed, and transferred
to PVDF membrane. The membrane was blocked with 2.5% (w/v) BSA in
PBS-Tween20, and incubated with the primary and secondary antibodies.
The secondary HRP-labeled antibodies were detected using enhanced
chemiluminescence reagents (Pierce) on a Kodak Imager (Kodak). Antibodies
used in this study were against LAT1 (Cosmo Bio), LAT3 (a kind gift
from Kunimasa Yan, Kyorin University, Tokyo, Japan), α-tubulin
(Santa Cruz), p-p70S6K, p70S6K, (Cell Signaling), UBE2C (Boston Biochem),
CDK1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam).
Horseradish peroxidase-conjugated donkey antimouse IgG, donkey antirabbit
IgG, and goat antimouse IgM were used as secondary antibodies (Millipore).
105 in 6-well plates, allowed to adhere overnight, before
incubation with DMSO, 10 mM BCH, 50 μM ESK242, or 50 μM
ESK246 for 6 h or 3 d. Cells were lysed by the addition of lysis buffer
(200 μL) with protease inhibitor Cocktail III (Bioprocessing
Biochemical) and 1 mM Na3VO4 (Sigma). Equal
protein (micro-BCA method; Pierce, IL) was loaded on 4–12%
gradient gels (Life Technologies), electrophoresed, and transferred
to PVDF membrane. The membrane was blocked with 2.5% (w/v) BSA in
PBS-Tween20, and incubated with the primary and secondary antibodies.
The secondary HRP-labeled antibodies were detected using enhanced
chemiluminescence reagents (Pierce) on a Kodak Imager (Kodak). Antibodies
used in this study were against LAT1 (Cosmo Bio), LAT3 (a kind gift
from Kunimasa Yan, Kyorin University, Tokyo, Japan), α-tubulin
(Santa Cruz), p-p70S6K, p70S6K, (Cell Signaling), UBE2C (Boston Biochem),
CDK1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam).
Horseradish peroxidase-conjugated donkey antimouse IgG, donkey antirabbit
IgG, and goat antimouse IgM were used as secondary antibodies (Millipore).