Figure 2. Expression of Kd and wild type or mutant tapasin in MF cell lines.
(A) The level of Kd and tapasin expressed in the indicated MF cell lines was determined by western blot analysis. Samples of cell lysates were electrophoresed on a 4→20% acrylamide Tris-glycine gel (for Kd) or on 10% acrylamide Tris-glycine gels (for tapasin and actin), transferred to membranes, and probed with the 64-3-7 mAb (for Kd), hamster anti-mouse tapasin mAb, or mouse anti-actin mAb. In the MF-Kd-SOL-ΔTsn cell line, soluble tapasin and tapasin Δ334-342 can be distinguished due to the smaller size of soluble tapasin. Noncontiguous lanes from the same gel are indicated by a dashed line. Data are representative of two experiments. (B) Stability of wild type and mutant tapasin. The indicated cell lines were incubated in the presence of 10 μg CHX/ml for 0, 4, or 8 h prior to lysis. Samples of the cell lysates were electrophoresed on a 10% acrylamide Tris-glycine gel, transferred to a membrane and probed with hamster anti-mouse tapasin mAb (upper panel). Proteins were then stripped, and the membrane was reprobed with mouse anti-actin mAb (lower panel). (C) Quantification of tapasin stability. Densitometry was performed on four different exposures with similar intensities of the tapasin and actin blots shown in (B). For each exposure, the relative signal of each band was determined by setting the band with the greatest signal to 100 in order to correct for the different intensities between exposures. Next, the average relative signal of each band from the four exposures was calculated. The average relative signal of tapasin was then normalized to the average relative signal of actin. Finally, the tapasin/actin ratio of cells treated with CHX for 0 h was set to 100% and used to calculate the percent tapasin/actin remaining at each time point. Error bars represent the standard deviation associated with the average relative signals from the four film exposures. Similar results were obtained on a separate SDS-PAGE/western blot of the samples.