Df4623

Immunofluorescence was performed for detecting METTL3, ZC3H13, and YTHDF2 expression in HCC and normal tissues. In brief, paraformaldehyde-fixed and paraffin-embedded tissue slices were cut into 5 μm thickness. The slices were incubated by anti-METTL3 (1 : 50; 15073-1-AP; Proteintech, Wuhan, China), anti-ZC3H13 (1 : 50; DF4623; AFFINITY, USA), and anti-YTHDF2 (1 : 50; 24744-1-AP; Proteintech, Wuhan, China) antibodies lasting 2 h. Following being washed, the slices were probed with ALexa Fluor 488-conjugated Affinipure goat antirabbit IgG (H + L) (SA00006-2; Proteintech, Wuhan, China) and DAPI (D9542; Sigma, USA). Then, the sections were mounted with glycerol and investigated under a fluorescence microscope.

Tissue and cell specimens were lysed by RIPA lysis reagent (Beyotime, China) on the ice for half hour and sonicated in an ice bath for 3 min. Following centrifugation at 4°C at 12,000 r/min for 10 min, the supernatant was harvested. The protein concentrations were calculated with BCA kit (Beyotime, China). Then, lysate was boiled with 5 × SDS loading buffer at 100°C for 5 min. Then, protein was separated by SDS-PAGE electrophoresis as well as transferred onto PVDF membranes (Millipore, Germany). Following being washed with TBST, the membranes were blocked by 5% milk/TBST lasting 1 h. Then, the membranes were probed with primary antibodies against METTL3 (1 : 1000; 15073-1-AP; Proteintech, Wuhan, China), ZC3H13 (1 : 1000; DF4623; AFFINITY, USA), YTHDF2 (1 : 1000; 24744-1-AP; Proteintech, Wuhan, China), or GAPDH (1 : 5000; ATA00013Rb; AtaGenix, Wuhan, China) at 4°C overnight. The membranes were washed by PBST for three times. Afterwards, the membranes were exposed to HRP-labeled goat antirabbit secondary antibody (SA00001-2; Proteintech, Wuhan, China) at room temperature for 1 h. The membranes were developed with luminescent buffer and investigated by ChemiDoc™ XRS + gel imaging system (Bio-Rad, Shanghai, China).