Plasmid construction and extraction
MrgC cDNA was cloned into pcDNA3.1-Myc-C plasmid and Ubiquitin or Ubiquitin mutant cDNA was cloned into pRK5-HA plasmid. Endotoxin-free plasmids were extracted for subsequent transfection using PureLink HiPure Plasmid Filter DNA Purification Kits (Invitrogen, USA).
Cell Culture And Transfection
Neuro-2a(N2a, American Type Culture Collection,USA)cells were grown in medium consisting of 90% DMEM (BI, Israel), 10% fetal bovine serum (Gibco, USA), 100 U/ml penicillin (Gibco, USA) and 100 µg/ml streptomycin (Gibco, USA) in a sterile incubator at 37°C, 95% O2 and 5% CO2.
Cells were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) with 3-4.5 µg plasmid per well. Cells were collected 48 hours post-transfection for subsequent experiments.
Western Blotting
Western blotting
Cells were washed 2 times with PBS and collected by adding M-PER (ThermoFisher) Mammalian Protein Extraction Reagent. The concentration of each protein sample was tested by BCA Protein Assay Kit. Protein samples were subjected to SDS polyacrylamide gel electrophoresis and then transferred to PVDF membranes. Primary antibodies were used anti-MrgC antibody (Mouse, 1:1000, Invitrogen), anti-Flag antibody (Rabbit ,1:1000,Cell Signaling Tech),anti-K48-linkage Specific Polyubiquitin antibody (Rabbit ,1:1000,Cell Signaling Tech), anti-K63-linkage Specific Polyubiquitin antibody (Rabbit ,1:1000,Cell Signaling Tech), anti-HA antibody (Rabbit ,1:1000,Cell Signaling Tech), anti-β-Actin antibody (Rabbit, 1:20,000, Bioss). The secondary antibodies were used sheep anti-rabbit (1:2500, Beyotime), sheep anti-mouse (1:20,000, Biosharp). Protein bands were detected by enhanced chemiluminescence (ECL, Vazyme, China), imaged with a cooled charge coupled device system (Tanon, China), and analyzed by ImageJ software.
Immunoprecipitation
Myc magnetic beads (1:20, Beyotime) were added to the cell lysate and incubated on a shaker at 4°C for 12 h. The immunoprecipitate was collected and repeatedly washed 3 times by adding 500 µl of lysis buffer. Add 30 µl 3×SDS sample buffer to the immunoprecipitate and heat in a 95°C water bath for 5 min. The samples were used for immunoblotting experiments.
Immunofluorescence
N2a cells were transfected with Myc-MrgC plasmid for 48h and incubated with Lyso-Tracker Red (Beyotime, 1:20,000) for 30min at 37°C. Cells were then fixed with 4% paraformaldehyde. The fixed cells were immunostained with anti-Myc antibody (Mouse, 1:4000, Cell Signaling Technology). The secondary antibody was Alexa 594-conjugated goat anti-mouse (1:1000, Invitrogen) and the nuclei were stained using DAPI (Abcam). Finally, immunostaining images were obtained using a laser-scanning confocal microscope (Olympus, Japan).
Statistical analysis
Statistical analysis was performed using SPSS 25.0 (IBM, USA). Data were expressed as mean ± standard error of mean. Immunoblot results were analyzed by one-way ANOVA followed by Dunnett's post hoc test. All tests were two-tailed and P < 0.05 was considered as the level of significance.