(A) Antigen binding ELISA. 100 ng of mouse p75NTRex-Fc (shown in dark blue), rat TrkAex-Fc, rat TrkBex-Fc, mouse NgRex-Fc, mouse or human APPex-Fc, N protein SL standard or BSA were immobilized in the plate for each well. 250 ng of each scFv was added after the antigen-coated plates were blocked with FCS for 1.5 hr. Bound scFvs were detected using anti myc-tag mAb (1∶500) and goat anti-mouse IgG HRP conjugated (1∶5,000). (B) The scFvs specifically recognize native p75NTR on PC12 cell surfaces. PC12 or HEK293T cells were stained with 250 ng of the p75NTR-specific scFvs (SH325-A11, SH325-B6, SH325-G7). Bound scFvs were detected by mouse anti-His₆ mAb (1∶100) followed by goat anti-mouse IgG F(ab′)² fragment APC conjugated (1∶200). The p75NTR surface expression on PC12 cells was determined by staining PC12 or HEK293T cells with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′)² fragment APC conjugated (1∶200). The blue histograms represent the mouse anti-p75NTR mAb (MLR2) or the p75NTR-specific scFvs staining PC12 cells (upper row) and HEK 293T cells (lower row). The white histograms represent the controls stained with goat anti-mouse IgG F(ab′)² fragment APC conjugated alone (MLR2 line) or αphOx scFv (other lines) followed by mouse anti-His₆ mAb and goat anti-mouse IgG F(ab′)² fragment APC conjugated. (C) Detection of denatured antigen (p75NTRex-mFc) by the p75NTR-specific recombinant antibodies in immunoblot. 250 ng of p75NTRex-mFc was denatured and blotted on a PVDF membrane. 1 µg of each p75NTR-specific recombinant antibody was used to stain the membrane for 1.5 hr. The bound antibodies were detected by goat anti-human IgG Fc antiserum AP conjugated (1∶2,000) for 1 hr at RT.

PC12 cells were transiently transfected with the p75NTR-specific ER retained intrabodies or αphOx-KDEL. Four days after transfection, the p75NTR surface expressions were determined with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′)² fragment APC conjugated (1∶200, B, D, F, H, and J). Cells stained with secondary antibody alone were served as control (A, C, E, G, and I). Overlay analysis for the p75NTR surface expression is shown in PC12 cells (K). The blue histograms represent the p75NTR surface expression of the PC12 cells expressing the p75NTR-specific ER retained intrabodies. The white histograms represent the p75NTR surface expression of the PC12 cells expressing the control αphOx-KDEL. The efficiency of knockdown p75NTR surface expression is indicated in percent. The picture of SH325-G7-KDEL is also shown in Figure 7 as 4 day time point result.

(A) Quantitative determination by immunoblotting. PC12 cells were transiently transfected with the constructs encoding the p75NTR-specific ER retained intrabodies. Four days after transfection, cell extracts were prepared and separated by SDS-PAGE. After immunoblotting, the membrane was incubated with mouse anti-His₅ mAb (1∶2,000) for 1 hr at RT and followed by goat anti-mouse IgG AP conjugated (1∶5,000) for 1 hr at RT. The control resulted from the cell extracts of untransfected PC12 cells. Intracellular expression levels of the p75NTR-specfic ER-intrabodies were calculated according to the scFv standard. (B) Quantification of intrabody production by densitometric analysis of gels as shown in (A). The error bars represent standard deviations calculated from three independent transfection experiments.

The SH325-G7-KDEL transfected PC12 cells from different time points (2–8 days) were sorted based on the EGFP-F fluorescence. Cells treated with 20 µg/mL of tunicamycin (Tun) or solvent alone (DMSO) were used as the positive or negative control, respectively. Cell extracts were prepared and blotted on a PVDF membrane. The membrane was incubated with rabbit anti-GRP94 (1∶1,000) and rabbit anti-GAPDH (1∶5,000) for 1 hr at RT. After 3× washing with PBST, the membrane was subsequently incubated with goat anti-rabbit IgG AP conjugated antibody (1∶5,000) for 1 hr at RT. GAPDH served as an internal control to ensure equal protein loading.

(A) PC12 cells transfected with the αphOx-KDEL construct. (B) PC12 cells transfected with the SH325-G7 construct. (C) PC12 cells transfected with SH325-G7-KDEL construct. (D) Percentage of differentiated PC12 cells in the cells transfected with different constructs (about 100 cells counted for each sample).

CMV: cytomegalovirus immediate-early promoter; VH: variable domain of heavy chain; VL: variable domain of light chain; KDEL: C-terminal ER retention signal; IRES: internal ribosomal entry site; EGFP-F: farnesylated enhanced green fluorescent protein; PolyA: BGH polyadenylation sequence.

100 ng of mouse p75NTRex-Fc fusion protein was immobilized in plates for each well. Serial diluted p75NTR-specific scFvs were added and incubated for 1.5 hr at 37°C. After 3× washing with PBST, the plates were incubated with mouse anti-p75NTR mAb (MLR2, 1∶5,000) for 1.5 hr at 37°C. The plate was washed 3× with PBST. The scFvs and mouse anti-p75NTR mAb (MLR2) were detected by mouse anti-His₅ mAb HRP conjugated (1∶5,000) and goat anti-mouse IgG HRP conjugated (1∶5,000) in corresponding plates.

NSC19 cells were transiently transfected with the p75NTR-specific ER retained intrabodies or the control αphOx-KDEL. Four days after transfection, the p75NTR surface expressions were determined with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′)² fragment APC conjugated (1∶200). The blue histograms represent the p75NTR surface expressions of the NSC19 cells expressing the p75NTR-specific ER retained intrabodies. The white histograms represent the p75NTR surface expressions of the NSC19 cells expressing the control αphOx-KDEL. The efficiency of knockdown p75NTR surface expression is indicated in percent.

PC12 cells were transiently transfected with SH325-G7-KDEL or the control αphOx-KDEL and harvested at different time points. The p75NTR surface expression levels were determined with mouse anti-p75NTR mAb (MLR2, 1∶200) followed by goat anti-mouse IgG F(ab′)² fragment APC conjugated (1∶200). The blue histograms represent the p75NTR surface expressions of the PC12 cells expressing SH325-G7-KDEL. The white histograms represent the p75NTR surface expressions of the PC12 cells expressing αphOx-KDEL. The efficiency of knockdown p75NTR surface expression is indicated in percent.

PC12 cells were transfected with the constructs of SH325-G7-KDLE, or the negative controls SH325-G7 or αphOx-KDEL. Six days after transfection, total RNA was extracted. The Bcl-xL mRNA expression, normalized to the housekeeping gene β-actin, was determined by real-time qRT-PCR. These results were then compared to the normalized Bcl-xL expression in untransfected PC12 cells. The error bars represent standard deviations from two independent experiments.