Figure 3.

ΔBAG2 impairs clearance of p-TAU, HSC70 binding and inhibits retinoic acid-induced differentiation in neuroblastoma cells. (A) Schematic representation of ZNF451-BAG2 fusion, the resulting truncated BAG2 is referred to as ΔBAG2. (B) BAG2 antibody was able to detect endogenous protein expression of ΔBAG2. Total cell lysates from SK-N-BE(1) and SH-SY5Y was immunoprecipitated with BAG2 antibody (Santa Cruz, catalog sc-390262) and protein levels were detected with the same BAG2 antibody. Whole cell lysate from SK-N-FI cells with lentivirally overexpressed ΔBAG2 was used as a positive control (ΔBAG2 OE). (C) Endogenous tumor ΔBAG2 protein expression is associated with high levels of phosphorylated TAU (p-TAU) in neuroblastoma tumors (NB) as detected by immunoblotting. Whole cell lysate from SK-N-FI cells with lentivirally overexpressed ΔBAG2 was used as a positive control (ΔBAG2 OE). Normal adrenal glands (AG). (D) Overexpression of the ΔBAG2 prevented clearance of pTAU by wt BAG2. SK-N-FI cells were transfected with p3XFLAG-CMV14-empty/BAG2/ΔBAG2/BAG2+ΔBAG2 for 48 h. Cells were harvested and proteins were extracted; whole-cell lysates were used to detect FLAG, p-TAU on Ser404, total TAU and ACTIN. (E, F) Wild type BAG2 but not ΔBAG2 binds to HSC70. FLAG-tagged proteins were immunoprecipitated from whole-cell lysates as prepared in (D) using Anti-FLAG M2 magnetic beads and eluted. Immunoprecipitated proteins were western blotting to detect HSC70, FLAG. (G–N) Constitutive lentiviral overexpression of ΔBAG2, but not wildtype BAG2 (backbone pLVX-EF1α-IRES-mCherry) inhibited RA-induced differentiation (6 days of treatment) in SK-N-FI cells. Data presented in (G) is western blot analysis of protein levels. Data in N is represented as mean of transduced cells with TUJ1⁺ neurites/total number of transduced cells ± SEM, each data-point represents this ratio in a single 10× microscopic field (n = 10–20). ***P< 0.001, one-way ANOVA with Tukey's multiple comparison test.