Sixteen hours after plating, media were switched to N2B27 to induce neural differentiation.
When cells reached a confluence of 60-80%, media were switched to serum-free media for drug treatment.
As HRG treatment resulted in the release of Ebp1 from ErbB3, we next determined whether HRG could regulate the association of endogenous Ebp1 with endogenous AR. LNCaP cells, growing in complete media, were switched into phenol-red free RPMI 1640 media with 1% CSS and R1881 (10−8 M) overnight.
To measure the association of endogenous AR and endogenous Ebp1, LNCaP cells, growing in complete media, were switched to phenol-red free RPMI 1640 containing 1% charcoal stripped calf serum (CSS) (Sigma) and 10−8 M R1881 (NEN, Boston, MA, USA) for 24 h.
After 24 h, the media were switched to DMEM phenol red free media with stripped serum (Hyclone, Logan, UT) for the cell proliferation assays, and DMEM phenol free and serum free for the western blot, phosphorylated receptor tyrosine kinase (pRTK) array and ELISA procedures.
At the end of the 14th day, inserts including islets were transferred into new 12-well plates and the culture media were switched to serum-free low glucose (L -DMEM (5.5 mmoL -DMEMntaining 0.5.5BSA for 2 h, washed twice with PBS, and then stimmol/ld by high glucontainingMEM (25 mmol/l) for 2 h.
