Nupage 4x

5–10 head samples were treated with lysis buffer (TBS1x, 150mMNaCl, IP 50x) and then homogenized and centrifuged 13500 rpm for 5 minutes. After selecting the supernatant we added NuPage 4x (Invitrogen by Thermo Fisher Scientific) and ß-mercaptoethanol 5%. Western blot analysis samples were run in a 4%–12% gradient SDS-PAGE for the detection of sHSP23 and sHSP26. After electro-blotted onto nitrocellulose 0.45 μM (GE Healthcare) 100V for 1 hour, we blocked the membranes in TBS-Tween-20 buffer with 5% BSA. We incubated the membranes overnight at 4°C in constant agitation with anti-Hsp23 antibody (1:1000) (Sigma-Aldrich S0821), anti-Hsp26 (1:1000) (Abmart) We visualized the antibody-protein interaction by chemo luminescence using IRDye Secondary Antibodies anti-mouse (IRDye 800CW, LI-COR), anti-rabbit (IRDye 680 RD, LI-COR) and developed with Odyssey equipment. Tubulin were used as a control.

The PMSA-derived products were digested with proteinase K (Roche) and subjected to electrophoresis and staining of total protein to assess the presence of proteinase K-resistant misfolded PrP (rec-PrP^res) in each sample. Briefly, 400 μl of each PMSA product were transferred to 1.5 ml Eppendorf tubes and proteinase K added to a final concentration of 25 μg/ml. The tubes were incubated at 42°C for 1 h and then centrifuged at 19,000 g at 4°C for 15 min. The supernatant was discarded carefully without disturbing the pellet and the pellet was washed with 500 μl of PBS and centrifuged again at 19,000 g for 5 min at 4°C. After removing all the PBS, the pellet was resuspended in 15 μl of loading buffer NuPage 4X (Invitrogen) diluted in PBS; at this point samples could be stored at −20°C until required. For electrophoresis, digested samples, together with non-digested controls, were boiled at 100°C for 10 min and loaded in 4–12% acrylamide gels (Invitrogen) for 1 h 20 min at 70 V for 10 min at 110 V for the next 10 min and 150 V for 1 h. The gel was then transferred to a glass bucket and total protein stained using BlueSafe (NzyTech) for at least 1 h at room temperature with gentle rocking.