Anti p62 18420 1 ap

A total of 3 × 10⁶ cells, previously washed in PBS, precipitated protein from supernatant cell culture, or animal tissues were lysed in 90 μL, 30 μL or 250 μL of RIPA buffers, respectively: RIPA Lysis Buffer with 100X protease inhibitors (Thermo Fisher Scientific). The lysates were incubated on ice for 1 h. The samples were centrifuged at 13,000 rpm for 15 min at 4 °C and the supernatant was collected in 4x Laemmli Sample Buffer (Bio-Rad, Hercules, CA, USA). Samples were loaded on a 7–14% gradient acrylamide gel (Bio-Rad), using 10× Tris/Glycine/SDS as a running buffer and the Bio-Rad electrophoresis system. The gels were transferred to PVDF membranes and blocked with 5% milk in TBS-Tween for 1 h. The membranes were incubated with different primary antibodies: anti-GAA (A7674-50, Abclonal Woburn, MA, USA), anti-p62 (18420-1-AP, Proteintech, Rosemont, IL, USA), anti-LAMP-1 (14-1071-82, Invitrogen Carlsbad, CA, USA) and anti-ERK1,2 (06-182A, Merck Millipore, Burlington, MA, USA) at 4 °C overnight and incubated with the secondary antibodies: goat anti-rabbit IRDye^® 800CV and donkey anti-rabbit IRDye^® 700CV (LI-COR) for 1 h. The membranes were scanned with the Odissey^® system (LI-COR, Lincoln, NE, USA).