Fig. 5: UBOX5 affects BIP half-life.

a Wild-type UBOX5 increases the half-life of BIP in the presence of thapsigargin-induced ER stress. Top left panel: Pulse chase of BIP with or without expression of UBOX5 in HEK293 cells. HEK 293 cells were transfected with UBOX5 or empty vector. 24 h later, cells were treated with a pulse of 0.25 μM Tharpsigargin for 2 hours, and cycloheximde (150 mM) was added. Cells were collected at indicated time points for immunoblotting. GAPDH was used as loading control. Positions of molecular weight standards are indicated on the left. Top right panel: The same experiment repeated with Tharpsigargin substituted with DMSO carrier control. Bottom panels: Quantitation of BIP band intensities normalized to GAPDH intensity for every indicated time point. Separate graphs were shown for cells treated with Tharpsigargin (Left) or DMSO control (Right). Intensities are shown as fold changes compared to normalized BIP intensity at t = 0 of pulse chase. Data are presented as mean values +/- standard deviation. Error bars indicate standard deviation; 3 biological replicates were used for quantitation. Source data are provided as a Source data file. b Variant UBOX5 and their effects on the half-life of BIP. Top: Pulse chase of BIP in the presence of wildtype UBOX5 or variant UBOX5 (D33N, K291R, R301Q, and S465C) in HEK293 cells. HEK 293 cells were transfected with wildtype UBOX or indicated variants. 24 h later, cells were treated with a pulse of 0.25 μM Tharpsigargin for 2 h, and cycloheximde (150 mM) was added at time = 0. Cells were then subsequently collected at indicated time points (in hours) for immunoblotting. GAPDH was used as loading control. UBOX5 expression was verified as indicated. GFP, expressed from a separate locus in the vector used, was also used to verify success of transfection. Bottom: Densitometric quantitation of BIP band intensities in cells expressing indicated UBOX5 variants, normalized to GAPDH intensity for every time point on the same blot. Intensities are shown as fold changes compared to normalized BIP intensity at t = 0 of pulse chase. Three biological replicates were analyzed. Error bars represent standard deviation. Source data are provided as a Source data file.