Figure 5
SG2NA is regulated at protein level by proteasomal degradation and its hyperphosphorylated form is more stable. NIH3T3 cells were treated with (A) 100 μM Okadoic acid alone and 10 μM MG132 and mixture of both (C) 10 μM MG132 alone (E) 10 mM LiCl (H) 25 μM PD98059, for various time points and western analysis was done with SG2NA and β-actin antibody. Graph representing quantitaive analysis of relative expression of SG2NA in time dependent manner upon their treatment is shown in (B), (D), (F) and (I) repectively. Confocal images of the cells treated with LiCl and PD98059 is shown in (G) and (J) respectively. Magnification- 60X, scale bar- 10 μm. (K) NIH3T3 cells were treated with various inhibitors as LiCl, PD98059 and LY294002 for 12 hrs and cell lysates were immunoprecipitated with SG2NA antibody followed by blotting with phosphoserine, phosphothreonine and SG2NA antibodies. Equal loading is shown by β-actin. (L) Graphs representing relative level of respective proteins with β-actin as loading control is shown. Error bars are the standard deviation of 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Full length blots for panel (A), (C), (E), (H) and (K) are shown in Supplementary Figure S1.
