Fig. 3

HSPA4 inhibits RNA m⁶A modification via stabilizing ALKBH5. A Dot blot analysis results of the total RNA of HSPA4-overexpressing or knockdown cells blotted with an anti-m⁶A antibody (upper). Methylene blue staining served as a loading control (bottom). B The protein levels of m⁶A methylation eraser (ALKBH5), writers (METTL3 and METTL14), and readers (YTHDF2 and YTHDC1) were determined by western blotting. β-Tubulin was used as a loading control. C The protein levels of HSPA4 and ALKBH5 in MKN45-HSPA4 and control cells transfected with siRNAs targeting ALKBH5. β-Tubulin was used as a loading control. D Total RNA was extracted from cells of C and m⁶A modification was examined by dot blotting (upper). Methylene blue staining served as a loading control (bottom). E Co-IP experiments were performed using either a HSPA4 antibody to pull down ALKBH5 or a ALKBH5 antibody to pull down HSPA4 in HGC27-HSPA4 cells, then proteins were examined. F, G The protein level of ALKBH5 was detected in HGC27-HSPA4 vs. control cells (F) and HSPA4 knockdown AGS (AGS-shHSPA4) vs. control cells (G) after cells were treated with CHX for different time. Relative protein levels of ALKBH5 were summarized in the line chart (right). (***, P<0.001; ****, P<0.0001)