Specific protein expression in cell lines was determined by western blot analysis or capillary electrophoresis as described before (6 (link)). The following antibodies were applied: p-MET (Tyr1234/1235) (Cell Signaling Technology (CST) 3077S; 1:250), MET (CST 8198S;1:1000), Mcl-1 (CST 94296S; 1:500), Bcl-2 (CST 15071S; 1:500), BIM (CST 2933S; 1:500), Bcl-xL (CST 2764; 1:500), Noxa (Millipore OP180; 1:500), β-actin (Sigma Aldrich A1978, clone AC15; 1:2,000). The HRP linked secondary antibodies were from Santa Cruz Biotechnology Inc. Western blot signals were detected by using a CCD – camera system (Azure C300 imager). For capillary electrophoresis (Wes instrument, Proteinsimple) the following antibodies were used: p-MET (Tyr1234/1235) (CST 3077S, 1:25), MET (CST 8198, 1:25), p-mTOR (Ser2448) (CST 5536S, 1:25), p-ERK (Thr202/Tyr204) (CST 4370S, 1:25), ERK (CST 4695, 1:25), mTOR (CST 2983, 1:25), p-CREB (Ser133) (CST 9198S, 1:25), CREB (CST 9197S, 1:25), PGC1α (CST2178, 1:25), Vinculin (Abcam ab129002, 1:200).
Protocol detail
Protein Expression Analysis by Western Blot and Capillary Electrophoresis
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Actin
Antibodies
Azure
Bcl 2
Capillary electrophoresis
Cell lines
Clone
Mtor
Pgc1α
Protein
Vinculin
Western blot
Western blot analysis
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Variable analysis
independent variables
- Cell lines
- Specific protein expression (p-MET (Tyr1234/1235), MET, Mcl-1, Bcl-2, BIM, Bcl-xL, Noxa, β-actin, p-mTOR (Ser2448), p-ERK (Thr202/Tyr204), ERK, mTOR, p-CREB (Ser133), CREB, PGC1α, Vinculin)
- Western blot and capillary electrophoresis protocols as described before (6)
- Antibody concentrations (1:250, 1:1000, 1:500, 1:2000, 1:25, 1:200)
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