(A) Preprolactin (pPL3f) and prolactin (PL3f) pulse-labeled with [35S]methionine and immunoprecipitated with anti-flag antibodies. T: translocation efficiency.
(B) Preprolactin (pPL3f) and prolactin (PL-3f and *PL3f) accumulation revealed with anti-flag antibodies. *PL: Phosphorylated prolactin in transit for secretion. *PL3f/SUP: relative levels of *PL in the medium.
(C) Same as (A) with pSEAP3f. 3fSEAP and p3fSEAP were precipitated with antibodies against SEAP (upper panel). p3fSEAP was precipitated with flag-tag antibodies (lower panel).
(D) TfnR-GFP was revealed with anti-GFP antibodies. preTfnR-GFP most likely represents the preprotein. *TfnR-GFP: modified mature protein. The preTfnR-GFP values were standardized to actin and normalized to shRNA(Luc)/G14 (n=2).
All experiments were done in 293T cells and equal amounts of cell extracts were loaded in each lane for Western blot analysis.