The separation efficiencies were checked by western blot assay (Supplementary Figure S1, available at Carcinogenesis Online) and total proteins of the three fractions were separated via 2-DE in triplicate, ensuring the spot reproducibility of more than 80%.
We first conducted western blot to check the AR protein levels in AR cells after treatment with RSV.
Each MEKK1 mutant was overexpressed in 293T cells in the highly expressing pApuro vector and analyzed via western blot to check for protein expression.
We also used Western blot to check the level of total Hsp27 protein between ALDH- and ALDH+ AS-B244 cells, which derived from ALDH+ BC0244 xenograft cells [ 14].
The precipitates were subjected to Western blots to check PKCε and CK18.
The protein loads on the blot were checked by staining with anti-ERK2 total antibody (item K-23 #sc-153, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a concentration suggested by the supplier.
The final supernatant was discarded and the inclusion body pellet was subjected to SDS PAGE, Coomassie brilliant blue staining and Western blotting for check of purity.
There have been very few measurements of rate constants reported for GSK-3, as the vast majority of studies use simple qualitative endpoint assays, for example western blotting to check for formation of phosphorylated substrates, to measure activity.
Next, we isolated G418-resistant clones in order to obtain cells that stably overexpressed the proteins, and we used RT-PCR and western blotting to check for efficient expression of MAWD and MAWBP (P < 0.001; Fig. 2a, b).
Levels of GSK3 β and p-GSK3 β in cells treated with celastrol (1 μM) or topotecan (1 μM) for 48 h were checked using western blotting assay.
