Horseradish peroxidase coupled secondary anti mouse or anti rabbit antibody

The Western blot method was previously described [9 (link)]. Antibodies against: mTOR (7C10) #2983, Phospho-mTOR (Ser2448) (D9C2) #5536, Phospho-mTOR (Ser2481) #2974, Phospho-p70 S6 kinase (Ser371) #9208, Phospho-p70 S6 kinase (Thr389) (108D2) #9234, p70 S6 kinase (49D7) #2708, phospho-S6 ribosomal protein (Ser235/236) (D57.2.2E) #4858, phospho-S6 ribosomal protein (Ser240/244) (D68F8) #5364, S6 ribosomal protein (5G10) #2217 (Cell Signaling Technology), N-cadherin #610920, Vimentin #550513, Phospho-FAK (pY397) #611722, FAK #610088 (BD Biosciences), and β-actin (A2228, SIGMA) were used to detect indicated proteins. Bands were visualized using horseradish peroxidase-coupled secondary anti-mouse or anti-rabbit antibody (Cell Signaling Technology). Immunoreactivity proteins were detected using chemiluminescence and images were captured with a ChemiDoc MP Imaging System (Bio-Rad Labs). To obtain quantitative results, immunoblots were scanned using SynGene Gene Tools version 4.03.0 (Synoptics Ltd Beacon House, Nuffield Road Cambridge, CB4 1TF, UK). Densitometry was used to normalize to the β-actin protein level. Presented are representative membranes of at least three independent experiments with similar results.

Samples for SDS-PAGE electrophoresis were prepared in the same way as previously described (Ciołczyk-Wierzbicka et al. 2012 (link)). Antibodies against Phospho-Bcl-2 (Ser70) (SH2) #2827, Phospho-Bcl-2 (Thr56) #2875, Bcl-2 (D55G8) 4223#, Bcl-xl (54H6) 2764#, Mcl-1 (D35A5) 5453# (Cell Signaling Technology), and β-actin (A2228, SIGMA) were used to detect the indicated proteins. Bands were visualized with the use of horseradish peroxidase-coupled secondary anti-mouse or anti-rabbit antibody (Cell Signaling Technology). Immunoreactivity of protein was detected with the use of chemiluminescence, and images were captured with a ChemiDoc MP Imaging System (Bio-Rad Labs). To obtain quantitative results, immunoblots were scanned with the use of SynGene Gene Tools version 4.03.0 (Synoptics Ltd. Beacon House, Nuffield Road Cambridge, CB4 1TF, UK). Densitometry was performed to normalize to β-actin protein level. Presented are representative membranes of at least three independent experiments.

Samples for SDS-PAGE electrophoresis were prepared as previously described (Ciolczyk-Wierzbicka et al. 2012 ). Antibodies against: Phospho-Bcl-2 (Thr56) #2875, Bcl-2 (D55G8) 4223#, Bcl-xl (54H6) 2764#, Mcl-1 (D35A5) 5453#, phospho-mTOR (Ser2448) (D9C2) #5536, phospho-mTOR (Ser2481) (cell signaling technology) and β-actin (A2228, SIGMA) were used to detect the indicated proteins. Bands were visualized using horseradish peroxidase-coupled secondary antimouse or antirabbit antibody (Cell Signaling Technology). The immunoreactivity of the protein was detected by chemiluminescence and images were captured with a ChemiDoc MP Imaging System (Bio-Rad Labs). To obtain quantitative results, immunoblots were scanned using SynGene Gene Tools version 4.03.0 (Synoptics Ltd Beacon House, Nuffield Road Cambridge, CB4 1TF, UK). Densitometry was performed to normalized β-actin protein level. Representative membranes of at least three independent experiments are presented.