Fig 2: BAG3 associates with NBR1 in neurons. C57Bl/6 mice at 15-month-old were sacrificed and the brain slices were used for immunostaining analysis. (A and B) Fluorescence images showed the colocalization of BAG3 with NBR1(A) or with SQSTM1(B) in mouse CA1 region of hippocampus. Scale bars, 20µm. (C) Graph shows the percentage of NBR1/SQSTM1 colocalized with BAG3 volume in total BAG3 positive volume and five mice in each group were used for study. Data were plotted as mean ± SEM. *, P<0.01. PL, Pyramidal layer; ML molecular layer. (D) Primary cortical neurons transduced with lentivirus expressing shBAG3 or a scrambled (Scr) version. The lysate of neurons was immunoprecipitated with a NBR1 antibody and immunoblotted for BAG3 to examine the association of BAG3 with NBR1. Black arrowhead points to the BAG3 band. (E) The lysate of transduced neurons was immunoprecipitated with a SQSTM1 antibody and blot for BAG3 to examine the association of BAG3 with SQSTM1. GAPDH was used as loading control.

Fig 3: Knock out of BAG3 in mouse brain increased the levels of phosphorylated and oligomeric MAPT. Cortices of 24-month-old control and BAG3Nes mice were collected for immunoblotting and immunostaining. (A) Cortical lysates from control and BAG3Nes brains were immunoblotted for phosphorylated MAPT (p-Ser262, p-Ser396/404, and p-Thr231). GAPDH is used as a loading control. (B-D) The graph shows the level of phosphorylated MAPT (p-Ser262, p-Ser396/404, and p-Thr231) normalized to GAPDH and relative to control. (E) Representative immunostaining of p-Ser396/404 MAPT (green) in the cortex of control and BAG3Nes. (F) Quantification of the percentage of cells with condensed staining of p-Ser396/404 MAPT. (G) Representative immunostaining of TOC1 MAPT (green) in the cortex of control and BAG3Nes. (H) Quantification of the fluorescence intensity in control and BAG3Nes mice. n = 3 for control and BAG3Nes. Scale bars, 20 µm. Data are shown as mean ± SEM. Statistical analysis was performed using the unpaired Student’s t test. *, p < 0.05; **, p<0.01.

Fig 4: Association of BAG3 and NBR1 is decreased in Alzheimer’s disease brain. (A) Representative immunostaining of BAG3 (red) and NBR1 (green) colocalization in human Alzheimer’s disease (AD) brain and age-matched control subjects. Scale bars: 10 µm. (B) Quantification of the colocalization between NBR1 and BAG3 based on volume. (C) Quantification of co-occurring BAG3 in NBR1, using Mander’s coefficient. (D) Representative immunostaining of BAG3 (red) and SQSTM1 (green) colocalization. (E) Quantification of the colocalization between SQSTM1 and BAG3 based on volume. (F) Quantification of co-occurring BAG3 in SQSTM1, using Mander’s coefficient. Six brains in each group were used for analysis. Data are shown as mean ± SEM using the unpaired Student’s t test; *p < 0.05; **p < 0.01.

Fig 5: BAG3 regulates the association of autophagy receptors with MAPT. (A) BAG3 Null HEK cells were transfected with MAPT, GFP-NBR1 together with BAG3 or control vector. Cell lysates were collected 48 hours post transfection and were immunoprecipitated with NBR1 antibody and immunoblotted with MAPT and NBR1. Five percent of the lysate were used as input control. (B) BAG3 Null HEK cells were transfected with MAPT, Myc-SQSTM1 together with BAG3 or control vector. Cell lysates were collected 48 hours post transfection and were immunoprecipitated with Myc antibody and immunoblotted for MAPT and Myc. Five percent of the lysate was used as input control. (C) FLAG M2 beads immobilized FLAG-NBR1 or BSA were incubated with purified His-MAPT together with GST or GST-BAG3. The precipitated samples were immunoblotted for His tag, BAG3 and NBR1. Five percent of the His-MAPT together with GST or GST-BAG3 used for pull down of both FLAG-NBR1 and Myc-SQSTM1 were used as input controls. (D) Myc beads immobilized Myc-SQSTM1 or BSA were incubated with purified His-MAPT together with GST or GST-BAG3. The precipitated samples were immunoblotted for His tag, BAG3 and Myc. (E) Myc beads immobilized Myc-SQSTM1 BSA were incubated with purified pre-formed fibrils together with or without the presence of GST-BAG3. The precipitated samples were eluted with Myc peptide. The eluted sample were split in half in reducing (Re) and non-reducing conditions (N-Re). The samples were immunoblotted with T22 and an antibody to SQSTM1. (F) FLAG M2 beads immobilized FLAG-NBR1 or BSA were incubated with purified pre-formed fibrils together with or without the presence of GST-BAG3. The precipitated samples were eluted with FLAG peptide. The eluted sample were split in half in reducing (Re) and non-reducing conditions (N-Re). The samples were immunoblotted for T22 and NBR1. The blots were represented images of three independent experiment.