β actin antibody

Cell lysates were collected using RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After heated at 75°C for 20 minutes, the samples were stored at −20°C. Before running, samples were sonicated and mixed with 2× Laemmli loading buffer (BIO-RAD, Hercules, CA, USA) and then heated at 95°C for 5 minutes. Each sample (30 μL) was loaded to 8% to 16% Tris-glycine gel (Invitrogen, Carlsbad, CA, USA), and electrophoresis was performed for 1 to 2 hours at 120 V. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and incubated with Odyssey blocking buffer (LI-COR Biotechnology, Lincoln, NE, USA) for 1 hour at room temperature. The membrane was incubated with primary antibodies diluted in blocking buffer with 0.01% Tween-20 at 4°C overnight. Antibodies for GRP78 (BIP; 1:1000) and PDI (1:500) were purchased from Cell Signaling Technology. β-Actin antibody (1:500) was purchased from BioLegend, Inc. (San Diego, CA, USA). Then, membranes were incubated with goat anti-mouse and goat anti-rabbit secondary antibodies (IRDye 680LT, IRDye 800CW; LI-COR Biosciences). The fluorescent signals were captured on an infrared imager (Odyssey Infrared Imager; LI-COR Biosciences).