Shb expression and cell proliferation of RIN-Shb and RIN-neo cells. (A) Exponentially growing cells were preincubated for 30 min with 20 µM N-acetyl-leu-leu-norleucinal, scraped and collected, and lysed in SDS sample buffer. The samples were boiled and equal amounts of protein from the RIN-Shb and RIN-neo cells were electrophoresed. After Western transfer, the blot was incubated with Shb-antibody and subjected to ECL detection as in Fig. 1. (B) Effects of serum on Shb expression. Equal amounts of RIN-neo cell were plated in four dishes and maintained in 10% serum for 24 hr. Two dishes (1,2) were cultured for an additional 18 hr in 10% serum whereas the other two (3,4) were maintained in 0.1% serum for 18 hr. The cells were collected and subjected to Western blot analysis for Shb protein expression as in Fig. 1. (C) Growth of RIN-Shb and RIN-neo cells in 10% serum. Cells (30,000) were plated after trypsinization and cultured in 1 ml RPMI 1640 + 10% FCS for the time points indicated. The cell numbers were counted in a Bürker chamber. Data for mean ± SEM for 6 to 8 experiments are given. (D) Growth of RIN-Shb and RIN-neo cells in 0.1% serum. Cells (30,000) were plated and cultured for the time points indicated in RPMI 1640 + 0.1% FCS and counted as in C. Data for mean ± SEM for 5 experiments are given. *p < 0.05 when compared with the value for RIN-Shb at 24 hr using a paired Students’ t-test.