FIG 6.

MG-132, rather than bafilomycin A1, partially rescues the steady-state expression of (N294A)S5-iHA. (A) HEK293 cells were transfected with either 50 ng of pBJ5-S5-iHA or pBJ5-(N294A)S5-iHA. Twenty-four hours after transfection, the culture medium was changed to include MG-132 (a proteasome inhibitor) or bafilomycin A1 (a lysosome inhibitor) at the indicated concentrations for 4 to 6 h. The cells were harvested, suspended in 1× TCEP reducing buffer, sonicated, and immunoblotted for SERINC5 (HA) and GAPDH. (B) The experiment shown in panel A was repeated, and the band intensities for S5-iHA and (N294A)S5-iHA from both experiments were quantified using ImageLab software (Bio-Rad). The values for the SERINC proteins were normalized to GAPDH and plotted using GraphPad Prism software; error bars are the standard deviations. The expression of wild-type S5-iHA and (N294A)S5-iHA in the presence of DMSO only were each set to 1.0.