Western Blot Analysis of Meq, Pu.1, v-rel, and GFP

Approximately 1 × 10⁶ HP8-ΔmiR-M4 cells and the control cells were collected and boiled with TruPAGE LDS sample buffer (Sigma) for 10 min. The samples were separated on a 4% to 12% TruPAGE precast gel, and the resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. Expression levels of Meq, Pu.1, v-rel, and GFP were detected using anti-Meq monoclonal antibody (MAb) FD7 (21 (link)), rabbit anti-SPIB polyclonal antibody (Aviva Systems Biology), anti-v-rel MAb HY87 (35 (link)), and GFP polyclonal antibody (SICGEN), respectively. The loading control used in all cases was α-tubulin (Sigma-Aldrich). After probing with primary antibodies was performed, the blots were incubated with secondary antibody IRDye®680RD goat anti-mouse IgG (Li-Cor) (for detection of Meq, v-rel, and α-tubulin), IRDye®800CW donkey anti-rabbit IgG (Li-Cor) (for detection of Pu.1), and IRDye®800CW donkey anti-goat IgG (Li-Cor) (for detection of GFP), and the results were visualized using Odyssey Clx (Li-Cor). For GFP detection, the PVDF membrane used for v-rel detection was stripped and reprobed with GFP antibody following the same procedure.