Horseradish peroxidase conjugated goat anti rabbit or anti mouse igg secondary antibodies

At 48 h post transfection of 5×10⁵ SH-SY5Y cells, total protein was extracted. Equal amounts (15 μg protein/lane) of proteins obtained from whole cell extracts of untransfected and Elk1 transfected samples were separated by SDS-PAGE and transferred onto nitrocellulose membrane by Trans-Blot Turbo Blotting System (Bio-Rad). Membrane was blocked in 5% skim milk powder/TBST (Tris buffered saline (TBS) containing 0.1% Tween 20) for 1 h at room temperature, and then incubated with the following primary antibodies at indicated dilutions overnight at 4°C; rabbit monoclonal His-tag antibody (1:1000, Cell Signaling Technology), mouse monoclonal spastin antibody (1:1000, Sigma), rabbit polyclonal katanin-p60 antibody (1:1000, ATLAS), mouse monoclonal p27 antibody (1:500, Santa Cruz), mouse monoclonal HuR antibody (1:500, Santa Cruz), mouse monoclonal β-Actin antibody (1:1000, Cell Signaling Technology), rabbit monoclonal GAPDH antibody (1:1000, Cell Signaling Technology), rabbit monoclonal β-tubulin antibody (1:1000, Cell Signaling Technology) in 5% skim milk powder/TBST. Membranes were then incubated with Horseradish peroxidase conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies (1:3000, Cell Signaling Technology) for 1 h at room temperature. Bands were visualized using Visualizer Western Blot Detection Kit (Millipore) and ChemiDoc Imaging System (Bio-Rad).

Total cellular proteins were extracted by utilizing RIPA lysis buffer (Thermo Fisher Scientific). Cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and proteins transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). These membranes were blocked with 5% bovine serum albumin (Geneall, Seoul, Republic of Korea) and incubated with primary antibodies against STAT3, p-STAT3, AMPK, p-AMPK, LC3B, p38, p-p38, p65, p-p65, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology). After primary antibody incubation, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies (Cell Signaling Technology). The protein bands were visualized using an enhanced chemiluminescent substrate (Thermo Fisher Scientific).