Immunoblot Protein Detection Protocol

Immunoblots were performed as previously described [6 (link),72 (link)]. Briefly, cells were harvested at the times indicated in lithium dodecyl sulfate (LDS) sample buffer followed by incubation at 95°C for 10 min. Denatured protein samples were run on NuPAGE 4–12% bis-Tris gels (Invitrogen). Proteins were transferred from the gel to a nitrocellulose or PVDF membrane. Membranes were blocked for 1 h in LI-COR blocking solution or in a solution of 5% (wt/vol) nonfat milk in PBS containing 0.1% Tween 20 (PBST). Membranes were incubated with primary antibody overnight at 4°C followed by 3x washes with PBST. Washed membranes were incubated in secondary antibody at room temperature for 1 h (IRDye 800CW Goat anti-Mouse IgG and IRDye 800CW Goat anti-Rabbit IgG for LI-COR; α-mouse IgG⁺HRP and α-Rabbit IgG⁺HRP, Cell Signaling 7076s and 7074s, respectively for film). Membranes were imaged with a LI-COR Odyssey imager (Lincoln, NE) or were incubated with SuperSignal West Pico or Femto chemiluminescent substrate (Thermo Scientific) and imaged by film or with a chemiluminescence imager.