Figure 4.

Effect of 17β‐E2 and NAC on JNK association with monomeric GSTP1‐1 form and GSTP1‐1 expression in MLO‐4Y cells. JNK association with GSTP1‐1 and GSTP1‐1 expression were detected in MLO‐Y4 cells cultured for 4 or 24 h in complete medium (C, control) or in serum‐free medium (S, starved cells). S were treated or not with 10 nm 17β‐E2 or 5 mm NAC as reported in Materials and methods. The negative control was performed in MLO‐Y4 cells cultured for 24 h in complete medium by immunoprecipitation with negative control mouse IgG (A). For detection of JNK bound to‐GSTP1‐1 or IgG (A and B), or GSTP1‐1 bound to JNK or IgG (A and C), or βActin bound to IgG (A) all immunoprecipitates of equal proteins (400 μg) were performed using anti‐GSTP1‐1 antibody or anti‐JNK antibody or IgG respectively, as reported in Materials and Methods. Western blot analyses were performed under non‐reducing conditions (without β‐mercaptoethanol) and membranes were probed with anti‐JNK (A and B) or anti GSTP1‐1 (C) antibodies and subsequently, after stripping, with anti‐GSTP1‐1 (A and B) or anti‐JNK (C) or anti‐βActin (A) antibodies, respectively. GSTP1‐1 expression (D) was detected by Western blot analysis performed under reducing condition. The normalized values with β‐Actin bands obtained by densitometric analysis are reported as mean percentage ± SEM relative to the respective C values in the bottom. The blots are representative of four experiments. *P ≤ 0.05 compared to the respective C values.