Long non-coding RNA MEG3 attends to morphine-mediated autophagy of HT22 cells through modulating ERK pathway

Gao, Shuibo; Li, Enyao; Gao, Haixia

Total proteins were separated from HT22 cells with the help of Cell Lysis Buffers (635656, Takara Biomedical Technology) including Protease Inhibitor Cocktail-ProteoGuard (635672, Takara Biomedical Technology). The concentration of total proteins was tested by TaKaRa CBA Protein Assay kit (T9300A, Takara Biomedical Technology). Then, proteins were electrophoresed on polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membrane (03010040001, Roche, Basel, Switzerland). Anti-OX1R antibody (sc-166111), anti-c-fos antibody (sc-271243), anti-t-ERK antibody (sc-514302), anti-p-ERK antibody (sc-81492), anti-t-PKC antibody (sc-8393), anti-p-PKC antibody (sc-377565), anti-Beclin-1 antibody (sc-48341), anti-LC3 antibody (sc-271625) and anti-b-actin antibody (sc-47778) were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PVDF membrane was incubated with primary antibodies for 12 h at 4 C. Subsequently, the PVDF membrane was incubated with Goat anti-rabbit (or anti-mouse) IgG-HRP (sc-2004, sc-2005) for 1 h at 20-25 C. After that, the PVDF membrane was placed into the Bio-Rad ChemiDoc TM XRS system (Bio-Rad Laboratories, Hercules, CA, USA) and coated with Pierce TM ECL Western Blotting (32109, Thermo Fisher Scientific, Waltham, MA, USA). The signals of proteins were recorded and the intensities of bands were calculated with the help of Image Lab TM software (Bio-Rad Laboratories).