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. 2010 Oct 20;24(12):2356–2365. doi: 10.1210/me.2010-0219

Figure 2.

Figure 2

A, Immunoprecipitation (IP) of endogenous PRLr from lysates from 293T cells treated with or without human PRL (purchased from the National Hormone and Peptide program and used at 100 ng/ml for 30 min) was carried out using anti-PRLr antibody (H-300, Santa Cruz) or naïve rabbit serum (NRS). Levels of PKM2 in whole-cell extracts (WCE) are also shown. B, MDA-MB-231 cells were infected with adenoviruses for delivery of human PRLr at increasing multiplicity of infection (MOI). WCEs were prepared and aliquots were resolved by SDS-PAGE and subjected to direct immunoblotting (IB) using anti-PRLr (N30, upper panel). Additional aliquots of the extracts were immunoprecipitated with N30 antibody and analyzed by IB using monoclonal anti-PRLr antibody from Invitrogen. NS, Nonspecific band. C, Lysates from the T47D cells (treated with or without 100 ng/ml of PRL for 15 min as indicated) were immunoprecipitated using either NRS or anti-PRLr N30 polyclonal antibody. Levels of PRLr and PKM2 in these reactions were detected by IB as indicated. Levels of PKM2, PRLr, and total and phosphorylated in the WCEs are also shown. IgG, Immunoglobulin heavy chain.