Figure 2: ERp44 regulates ERAAP levels.

A. To confirm the results of the CRISPR KO screen, BJAB ERAAP dsRed cells (clone B2) were transfected with two gRNA targeting ERp44 or one gRNA targeting ERAAP as a control. ERAAP levels in ERp44 KO cells were measured by western blot. Representative blot showing ERAAP and ERp44 protein levels in untransfected BJAB ERAAP dsRed (WT) and KO cells. GAPDH is shown as a loading control. B. Histogram showing ERAAP dsRed levels in BJAB ERAAP dsRed (blue histogram) and ERp44 KO cells (orange histogram). Parental BJAB cells are shown as a control (grey filled histogram) C. ERAAP secretion from ERp44 KO cells was measured by LAP assay. ERAAP was immunoprecipitated from the supernatants of BJAB ERAAP dsRed and BJAB ERAAP dsRed ERp44 KO cells. Beads containing ERAAP were incubated with LpNA substrate and LAP activity was measured 8 h after addition of the substrate by measuring optical density (OD) at 410 nm. Background activity coming from media was subtracted from both conditions. Shown is the fold change in LAP activity relative to BJAB ERAAP dsRed (WT) cells. Data represents mean± SD from three independent experiments. Unpaired two-tailed T-test was performed. ** p value < 0.01.