Figure 7

HMGB1 promoted Beclin-1-PI3K-III core complex formation through the MEK/ERK1/2 pathway. (A) SPC-A1 cells were transfected with pcDNA3.1-HMGB1, PI3K-III siRNA or both for 48 h. Total cell lysates were subjected to western blot analysis of LC3, p62, p-ERK1/2 and GAPDH (as a loading control). (B, E) SPC-A1 cells were pretreated with PD098059 (20 μg/ml, 45 min), followed by transfection with control or pcDNA3.1-HMGB1. Whole cell lysates were analyzed by (B) western blot against LC3, p62, and p-ERK1/2 and (E) immunoprecipitation (IP) as described in Materials and methods. (C) SPC-A1 cells were transfected with pcDNA3.1-HMGB1, MEK siRNA or both for 48 h. Total cell lysates were examined by western blot analysis using specific antibodies against LC3, p62, MEK and p-ERK1/2. (D, F) SPC-A1 cells were transfected with control or HMGB1 shRNA, and then infected with the constitutively active MEK1 construct, MEK1-DD. Total cell lysates were analyzed by (D) western blot for LC3, p62 and p-ERK1/2 and (F) IP. The blots shown are representative of three separate experiments in which similar results were observed.