Fig. 4

Autophagic suppression by Hcy is the cause of ER stress in Hyperhomocysteinemic neurons. Representative immunoblots of same cell lysates showing a time dependent suppression of autophagy (A) and an activation of ER stress (B) by Hcy in primary neurons. β-actin was used as a loading control. Densitometric values in the blots represent the ratio of respective protein signal to β-actin signal normalized to control. Representative immunoblots of ER-stress marker proteins showing increased expression by autophagic inhibitor BafA1 (20 nM) treatment (C) and decreased expression by autophagic activator RAPA (200 nM) treatment (D) in Hcy-treated SK-N-SH cells. β-actin was used as a loading control. Densitometric values in the blots represent the ratio of respective protein signal to β-actin signal normalized to untreated control. E Immunoblot confirming higher expression of ATG7 protein in a stable neuroblastoma cell line overexpressing ATG7. GAPDH was used as a loading control. F Representative immunoblots showing decreased expression of ER-stress markers after Hcy-treatment in ATG7 overexpressing (constitutively activated autophagy) B35 cells compared to vector control cells. β-actin was used as a loading control. Densitometric values in the blots represent the ratio of respective protein signal to β-actin signal normalized to control. Concentration of Hcy was 5 mM. Data are shown as mean ± SEM with n ≥ 3