Journal: Neural Regeneration Research
Article Title: Salsolinol as an RNA m 6 A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy
doi: 10.4103/NRR.NRR-D-23-01592
Figure Lengend Snippet: m 6 A RNA methylation regulates YAP1 expression in vitro and in vivo . (A) m 6 A modification at the 1510 and 1520 sites of rat Yap1 mRNA after FTO or ALKBH5 was knocked down. (B) m 6 A modification at the 2156 site of the rat Yap1 mRNA after FTO or ALKBH5 was knocked down. (C) m 6 A modification at the 2795 site of the rat Yap1 mRNA after FTO or ALKBH5 was knocked down. (D) YAP1 protein expression did not change in PC12 cells after knocking down FTO. (E) YAP1 protein expression decreased in PC12 cells after knocking down ALKBH5. (F) YAP1 protein expression remained unchanged In the striatum of the rat brain after FTO was knocked down and decreased after ALKBH5 was knocked down. (G) YAP1 protein expression remained unchanged in the midbrain of the rat after FTO was knocked down and decreased after ALKBH5 was knocked down. Data were normalized by the control group. Data are expressed as the mean ± SD ( n = 3/group). * P < 0.05, ** P < 0.01, **** P < 0.0001, vs. Scr group (Student’s t -test). ALKBH5: alkB homolog 5; FTO: fat mass and obesity-associated protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; IP: immunoprecipitation; m 6 A: N 6 -methyladenosine; Scr: scramble; shALKBH5: short hairpin RNA of alkB homolog 5; shFTO: short hairpin RNA of fat mass and obesity-associated protein; YAP1: Yes-associated protein 1.
Article Snippet: The membrane was blocked with 5% skimmed milk and incubated with mouse anti-FTO monoclonal antibody (1:1000, Abcam, Cat# ab92821, RRID: AB_10565042), rabbit anti-ALKBH5 monoclonal antibody (1:1000, Abcam, Cat# ab195377, RRID: AB_2827986), rabbit anti-METTL3 monoclonal antibody (1:1000, Abcam, Cat# ab195352, RRID: AB_2721254), mouse anti-YAP1 monoclonal antibody (1:8000, Proteintech, Chicago, IL, USA, Cat# 66900-1-Ig, RRID: AB_2882229), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:100,000, Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), rabbit anti-YTH domain-containing family protein 1 (YTHDF1) polyclonal antibody (1:2000, Proteintech, Cat# 17479-1-AP, RRID: AB_2217473), rabbit anti-YTH domain-containing family protein 2 (YTHDF2) polyclonal antibody (1:5000, Proteintech, Cat# 24744-1-AP, RRID: AB_2687435), mouse anti-YTH domain-containing family protein 3 (YTHDF3) monoclonal antibody (1:1000, Santa Cruz Biotechnology, Texas, CA, USA, Cat# sc-377119, RRID: AB_2687436), rabbit anti-p62 polyclonal antibody (1:1000, MBL, Tokyo, Japan, Cat# PM045, RRID: AB_1279301), rabbit anti-light chain 3 beta (LC3B) polyclonal antibody (1:1000, Cell Signaling Technology, Boston, MA, USA, Cat# 2775, RRID:AB_915950), and rabbit anti-phospho-YAP1 (Ser127) (1:1000, Cell Signaling Technology, Cat# 13008, RRID: AB_2650553) at 4°C overnight.
Techniques: Methylation, Expressing, In Vitro, In Vivo, Modification, Control, Immunoprecipitation, shRNA
