The activation of Akt and ERK pathway was analyzed by western blotting following the steps described before using the primary antibody against phospho-Akt (p-Akt, 1 : 500, Cell Signaling), Akt1 (1 : 100, Santa Cruz), phosphor-ERK (p-ERK, 1 : 2000, Cell Signaling), ERK2 (1 : 200, Santa Cruz), and β-actin (1 : 10000, Sigma) [ 25].
With the information of the roadside obtained in the processing step described before, it is possible to restrict vehicle detections and tracking only to the well determined road areas.
Although the method described before has a well-defined set of steps to follow, what is stipulated in Step 3 turns out to be very open or intuitive.
It should be noted that individually most of the QC steps described have been used before in other CNV studies (10, 44, 67, 68 and Supplementary Material, Methods S4), although not in combination, or with as extensive calibration and validation.
In separate experiments, cells were first incubated for 1 h with RPMI, MEK inhibitor (5 µM), JNK inhibitor (20 µM) or p38 inhibitor (1 µM) before performing the steps described above.
Follow the steps described above.
For distinguishing specific from non-specific products and primer dimers, a melting curve was obtained after amplification by holding the temperature at 70°C for 30 s followed by a gradual increase of temperature to 98°C at a rate of 0.2°C/s, with the signal acquisition mode set at step, as described before (Woo et al, 1998).
However, visualization cannot quantify expression levels nor find global patterns; the steps that were described before provide the systematic genome-wide quantification of information in RNA-seq experiments.
The following steps were performed as described before.
Taken together, these data show that the drug combination of BA plus doxo activated the identical downstream signaling steps that have been described before for BA or doxo alone.
The following morning, slides were rinsed in ddH2O 3X before proceeding to the blocking step described above.
