Figure 4. Interactions of SNCA-HMGB1, HMGB1-BECN1, and BECN1-BCL2. (A) Immunoprecipitation (IP) of overexpressed HA-SNCA and endogenous BECN1 with endogenous HMGB1 and immunoprecipitation of endogenous BECN1 with endogenous BCL2 in iPC12 cells treated with 2 μg/ml Dox for 24 h. Experiments were performed 3 times with similar results and the representative blots were shown. (B) Ratio of HMGB1-BECN1 and BCL2-BECN1 interaction. The levels of immunoprecipitated BECN1 were normalized to the corresponding levels of BECN1 in whole cell lysates. Data are presented as the mean ± SD from 3 independent experiments. *P < 0.05 vs. uninduced control. (C) Immunoprecipitation of SNCA and endogenous BECN1 with endogenous HMGB1 in cytosolic (Cyt.) and nuclear (Nuc.) fractions of PC12 cells transfected with GFP-SNCA. 0.5 mg cytosolic protein and 1 mg nuclear protein were used for immunoprecipitation, respectively. In parallel, 10 μg cytosol protein and 20 μg nuclear protein were loaded as inputs. ACTB and LMNB1 were used as loading control of cytoplasmic and nuclear fraction respectively. Experiments were performed 3 times with similar results and the representative blots were shown.