Total cell lysates (20 µg) were resolved by 10% SDS‐PAGE, transferred to nitrocellulose membranes and immunoblotted with antibodies to BCAT1 (Abcam), Ras, phosphor (p)‐BRaf (Ser445)/total (t)‐BRaf, p‐MEK (Ser217/221)/t‐MEK, p‐ERK (Thr202/Tyr204)/t‐ERK and p‐Akt (Ser473)/t‐Akt (Cell Signaling). β‐Actin served as the loading control (Cell Signaling). Immunoreactive bands were identified using an enhanced chemiluminescence reaction (Perkin‐Elmer) and visualized by autoradiography.
Bcat1
Manufactured by Abcam
Sourced in United Kingdom
BCAT1 is a protein-coding gene that is involved in the metabolism of branched-chain amino acids. The encoded enzyme catalyzes the reversible transamination of the branched-chain amino acids leucine, isoleucine, and valine.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence reaction, by PerkinElmer (1 mentions)
P erk thr202 tyr204 t erk, by Cell Signaling Technology (1 mentions)
P akt ser473 t akt, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
Lookout mycoplasma pcr kit, by Merck Group (1 mentions)
3 protocols using bcat1
1
Western Blot Analysis of Cell Signaling
2
Western Blot Protein Analysis
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Total proteins were extracted from the RCC cell lines, WB assay was performed after the detection of protein concentration. 20 μg of samples were separated on a 10% SDS-PAGE gel, then transferred to a PVDF membrane and blocked for 1 hour at room temperature. The membranes were incubated with primary antibodies (BCAT1 concentration, 0.5 μg/mL; GAPDH dilution rate, 1:500; Abcam, UK) at 4° C overnight. The next day, the membranes were incubated with the secondary antibody (Abcam; dilution rate, 1:2000) at 24° C for 1 h. Signals of targeted proteins were detected using an enhanced chemiluminescence detection system.
3
Metabolic Adaptation of Pancreatic Cancer Cells
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Reagents. Antibodies for western blotting were from Abcam: BCAT1 (ab107191), GOT2 / FABP-1 (ab171739); BD Biosciences: GS / GLUL (BD-61057); Cell Signaling Technology 8988T) and were authenticated by STR profiling at ATCC. All cell lines tested negative for mycoplasma using LookOut Mycoplasma PCR Kit (Sigma, MP0035). All cells were maintained at 37°C in a humidified incubator with 5% CO2 and were grown in media supplemented with 10% dialyzed fetal bovine serum (Sigma-Aldrich F0392). For adaptation to "low glucose, low glutamine" (L-L), "low glucose-high glutamine" (L-H), or "high glucose-low glutamine" (H-L), PDA cells were cultured in RPMI 1640 medium lacking both glucose and L-glutamine (US Biological, R9011-01) that was supplemented with either 0.5 mM glucose and 0.1 mM glutamine for L-L; or with 0.5 mM glucose and 2mM glutamine (L-H); or with 11mM glucose, 0.1mM glutamine (H-L). Non-adapted cells were cultured in the same RPMI 1640 medium that was however supplemented with 11mM glucose and 2 mM glutamine, and was termed "high glucose, high glutamine" (H-H) medium. Glucose (A2494001) and glutamine (25030-81) were from Thermo Fisher Scientific.
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