Cell Lysate Preparation and Immunoblotting

The method used for cell lysate preparation and immunoblotting were described previously (Pathak et al., 2013 (link)). Briefly, cell lysates were prepared by the addition of SDS-PAGE sample reducing buffer to cells after washing twice with PBS, followed by heating at 95°C for 10 min. Immunoblotting was performed using anti-phospho ERK1/2 (D13.14.4E) (1:1,000 dilution), anti-phospho-p38 (D3F9), anti-phospho-JNK (81E11), and anti-ERK1/2 (137F5) (1:1,000 dilution) antibodies from Cell Signaling Technology. Anti-phospho-c-Fos (Thr325) antibody was from Bioss antibodies. An anti-actin antibody (Thermo Scientific) (1:5,000 dilution) was used to confirm equal loading. The secondary antibodies included goat anti-rabbit IgG and goat anti-mouse IgG conjugated to IRdye800CW (Li-COR) (1:10,000 dilution). Band intensities were quantified using the ImageJ software, and actin was used to normalize the total amount of protein loaded in each well.