Figure 4.
Figure 4. Cellular and biochemical phenotype of the CD4tl-λC upon inactivation of CHIP. (A) Effects of dominant-negative (DN) E3 and E2 enzymes on the PM stability of CD4tl-λC were measured in transiently cotransfected HEK293 cells and monitored by cell surface ELISA after 30-min chase at 37°C. Fig. S3 D depicts the CD4tl-λ PM stability (**, P < 0.01; n = 5). The dotted line indicates the chimera cell surface turnover in mock-transfected cells. (B) CHIP ablation was accomplished by lentiviral shCHIP in Flp-In T-Rex HEK293 cells expressing CD4tl-λC and verified by immunoblotting. CHIP-myc overexpression was detectable in the shCHIP cells. CHIP depletion had an adverse effect on tetracycline transactivation, reducing the induction level of CD4tl-λC in shCHIP cells. (C) Ubiquitination level of the PM CD4tl-λC was probed by denaturing cs-IP and immunoblotting. Three times more shCHIP cells were used. (D) Stability of internalized CD4tl-λC was measured by labeling the PM-resident chimera with anti-CD4 Ab at 4°C and chasing for 0–1 h at 37°C in shNT and shCHIP cells. Ab–chimera complexes were precipitated and immunoblotted. (E) The PM turnover of the CD4tl-λC was measured by cell surface ELISA in control, shCHIP, and CHIP-myc–transfected cells. (F) The CD4tl-λC and CD4cc-UbAllRΔG (UbRΔG) PM stability was determined after 1-h chase in shCHIP cells coexpressing the CHIP-myc variant. (G) The internalization rate of CD4tl-λC was monitored in the absence or presence of CHIP-myc at 37°C by Ab uptake in shCHIP cells for 5 min. Endocytosis of tetrameric CD4cc-UbAllRΔG (UbRΔG) and transferrin (Tf) was measured for 2.5 min. (H) Mean vesicular pH of internalized cargo was determined by FRIA in small interfering NT and small interfering CHIP (siCHIP) cells as described in Materials and methods. Anti-CD4 Ab and FITC-Fab were bound on ice, and FRIA was performed after 1-h chase at 37°C. NT, nontargeted siRNA. The lysosomal targeting of CD4tl-Ub, CD63, and dextran was not influenced by shCHIP. Molecular mass is given in kilodaltons. IB, immunoblot. The data shown represent means ± SEM.

Cellular and biochemical phenotype of the CD4tl-λC upon inactivation of CHIP. (A) Effects of dominant-negative (DN) E3 and E2 enzymes on the PM stability of CD4tl-λC were measured in transiently cotransfected HEK293 cells and monitored by cell surface ELISA after 30-min chase at 37°C. Fig. S3 D depicts the CD4tl-λ PM stability (**, P < 0.01; n = 5). The dotted line indicates the chimera cell surface turnover in mock-transfected cells. (B) CHIP ablation was accomplished by lentiviral shCHIP in Flp-In T-Rex HEK293 cells expressing CD4tl-λC and verified by immunoblotting. CHIP-myc overexpression was detectable in the shCHIP cells. CHIP depletion had an adverse effect on tetracycline transactivation, reducing the induction level of CD4tl-λC in shCHIP cells. (C) Ubiquitination level of the PM CD4tl-λC was probed by denaturing cs-IP and immunoblotting. Three times more shCHIP cells were used. (D) Stability of internalized CD4tl-λC was measured by labeling the PM-resident chimera with anti-CD4 Ab at 4°C and chasing for 0–1 h at 37°C in shNT and shCHIP cells. Ab–chimera complexes were precipitated and immunoblotted. (E) The PM turnover of the CD4tl-λC was measured by cell surface ELISA in control, shCHIP, and CHIP-myc–transfected cells. (F) The CD4tl-λC and CD4cc-UbAllRΔG (UbRΔG) PM stability was determined after 1-h chase in shCHIP cells coexpressing the CHIP-myc variant. (G) The internalization rate of CD4tl-λC was monitored in the absence or presence of CHIP-myc at 37°C by Ab uptake in shCHIP cells for 5 min. Endocytosis of tetrameric CD4cc-UbAllRΔG (UbRΔG) and transferrin (Tf) was measured for 2.5 min. (H) Mean vesicular pH of internalized cargo was determined by FRIA in small interfering NT and small interfering CHIP (siCHIP) cells as described in Materials and methods. Anti-CD4 Ab and FITC-Fab were bound on ice, and FRIA was performed after 1-h chase at 37°C. NT, nontargeted siRNA. The lysosomal targeting of CD4tl-Ub, CD63, and dextran was not influenced by shCHIP. Molecular mass is given in kilodaltons. IB, immunoblot. The data shown represent means ± SEM.

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