Figure 4

Total ubiquitination Western blot (WB) analysis of cultured PBMCs from ALS patients and controls in the presence or absence of added-back serum for 16 hours and treated or not with proteasome inhibitor MG132 for 1.5 hr. Comparison of PBMCs from one healthy control and one ALS patient incubated or not in the presence of added-back matched autologous serum is shown in (a). Semi-quantitative Western blot analysis was performed to measure the accumulation of high molecular weight (HMW) ubiquitinated protein species in PBMCs that were prepared the same day from one healthy control and one ALS patient (WB1). A signal (S) to noise (N) ratio (S/N) was determined with ImageJ program by comparing the integrated density of two areas consistently stained throughout the membrane and visually contrasting the accumulation of HMW ubiquitinated protein species (WB1). ALS patient serum exacerbates the effects of MG132 on total ubiquitination and accumulation of HMW ubiquitinated species, while serum from healthy control mitigates these effects. Comparison of PBMCs from ALS patients (n = 5) and healthy controls (n = 4) incubated in the presence of added-back matched autologous serum is shown in (b). PBMCs obtained at different times from additional ALS patients (n = 5) and healthy controls (n = 4) show similar result (WB2 and WB3).