Plakoglobin binds to SOX4 in LNCaP cells. A. Whole cell lysates prepared from LNCaP cells transfected with pREP4-BLRPwt-HASOX4-IRES-BirA-XL9 or vector only were purified using an streptavidin-magnetic beads, and affinity-purified Vector- and SOX4-complexes were visualized by silver staining. SOX4 protein is indicated by an arrow. The molecular weight markers are as shown on the left. B. Amino acid sequence of plakoglobin with trypsinized peptides from LC-MS/MS analysis indicated in bold and underlined. C, pREP4-BLRPwt-HASOX4-IRES-BirA-XL9 and vector control transfected LNCaP cells. SOX4-associated plakoglobin were analyzed by Western blot with antibodies indicated. Equivalent amounts of the Vector-purified fractions were used to confirm specificity. 5% of transfected LNCaP whole cell lysate was used as Input. D, endogenous plakoglobin IPs were analyzed for transfected SOX4 by Western blot with anti-HA 16B12 mAb. E, endogenous plakoglobin IPs were analyzed for endogenous SOX4 in untransfected LNCaP cells. F, G, endogenous plakoglobin IPs were analyzed for endogenous SOX4 in primary human keratinocytes and breast cancer cell line, MDA-MB-231, respectively. H, I, HASOX4 IPs were analyzed for endogenous plakoglobin in stably-expressed SOX4 PC3M and LNCaP cell lines. F-I, whole cell lysates were harvested and treated with DNase I for 1 hr at room temperature prior to immunoprecipitation.
Lai et al. BMC Cell Biology 2011 12:50 doi:10.1186/1471-2121-12-50