Rabbit anti human akt c67e7

After treated with CXCL8 or transfected with CXCL8 siRNA/non-specific siRNA for 48 h, the cells were lysed by RIPA (Beyotime, Haimen, China) supplemented with protease inhibitor (Beyotime, Haimen, China). Electrophoresis was carried out on sodium dodecyl sulfate polyacrylamide gel according to the standard of 30 ug/lane. After electrophoresis, the protein was transferred to polyvinylidene fluoride membrane. The primary antibody was incubated overnight at 4 °C and the secondary antibody labeled with horseradish peroxidase was incubated at room temperature for 1 h. Western Blot bands were detected by emitter coupled logic (ECL) solution (Beyotime, Haimen, China). The image was semi quantitatively analyzed by Image J software.
Primary antibodies were as follows: rabbit anti-human p-AKT (Ser473) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-human AKT (C67E7) (Cell Signaling Technology, Danvers, MA, USA), and anti-human LSECtin (ab181196) (Abcam, Cambridge, UK). Primary antibodies were as follows: Rabbit anti human p-AKT (ser473) (cell signaling technology, Danvers, MA, USA), Rabbit anti human AKT (c67e7) (cell signaling technology, Danvers, MA, USA), and Rabbit anti human LSECtin (ab181196) (Abcam).

Primary antibodies were as follows: mouse anti-human TrkB, mouse anti-human ALK, mouse anti-human caspase-3 and cleaved caspase-3, mouse anti-human Bcl-2, mouse anti-human Bax, mouse anti-human NF-κB (p65) (Abcam, Cambridge, UK), rabbit anti-human pAkt (Ser473), rabbit anti-human pALK (Tyr1507), rabbit anti-human Akt (C67E7), rabbit anti-human p38 MAPK (D13E1), rabbit anti-human pERK1/2 (Thr202/Tyr204), and rabbit anti-human ERK1/2 (137F5) (Cell Signaling Technology, Danvers, MA, USA). GAPDH (detected as a loading control), mouse anti-human Midkine and all secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The primary and secondary antibodies were used at 1/2000 dilution. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Sigma-Aldrich) with exposure to X-ray film. The resultant film images were analyzed by Quantity One analysis software (Bio-Rad, Hercules, CA, USA). The results were quantified by normalization of three separate experiments to GAPDH values.

At 48 h after RNA transfection, Cells were lysed using a protein extraction reagent containing protease inhibitor (Beyotime). Total protein concentrations were determined using a BCA protein assay reagent (Beyotime). Then, western blot was implemented as previously described [14 (link)]. The primary antibodies used in this study were as follows: mouse anti-human E-cadherin (Abcam), mouse anti-human vimentin (Abcam), rabbit anti-human p-AKT (Ser473) (Abacm), and rabbit anti-human AKT (C67E7) (Cell Signaling Technology) and GAPDH (Beyotime). The semi-quantitative analysis of the western blots was conducted using Image J software.