VSMC from three old rats were sub-cultured overnight and on the second day, cells were pre-treated with 20μM of AG1024, LY294002, or TCN for 30 min and then grown for 3 days in medium containing 5 mM or 12.5 mM glucose with 20 μM of each inhibitor, respectively. A. Examples of Western blots showing P-ERK1/2, ERK1/2 and PCNA in total proteins, c-Fos in nuclear proteins. Each includes the same blot of β-actin as loading control. B. Changes of P-ERK1/2, c-Fos and PCNA in AG1024-, LY294002-, and TCN-treated cells summarized as a percentage of 5 mM glucose control (mean ± SE). The individual of p values are described in the text and the * indicates p ≤ 0.05 for comparisons between cells treated with inhibitors and corresponding controls. C. An example of Western blots comparing the effect of AG1024 with LY294002 on P-JNK changes in total proteins.