Anti trpm2

According to reported methods [17 (link)], the prepared membranes were obtained through collection, separation and transmembrane, and then incubated with rabbit primary antibodies for overnight at 4 °C. Antibodies included anti-TRPM2 (1:500, Thermo Fisher Scientific), anti-ox-CaMKⅡ (1:500, 07–1387, EMD Millipore), anti-p-ERK(Tyr204) (1:500, AF1014, Affinity), anti-p-MEK (Thr292) (1:500, AF3383, Affinity) and anti-GAPDH (1:1000, AF7021, Affinity). After washed with TBS-0.01 % Tween 20 for 3 times, the membranes were incubated with the secondary antibody (1:3000, S0001, Affinity) for 2 h at 25 °C. An enhanced chemiluminescence reagent (D085075, Bio-Rad, China) was used to visualize the bands.

Cells were fixed with 4 % paraformaldehyde for 30 min and blocked with TBS-T containing 5 % BSA for 60 min. After washing, the cells were incubated with rabbit primary antibodies, anti-TRPM2 (1:200, Thermo Fisher Scientific) and anti-ox-CaMKⅡ (1:200, 07–1387, EMD Millipore), overnight at 4 °C without light. After washing, the cells were incubated with Cy3-labeled goat anti-rabbit IgG (H + L) antibody (1:200, S0008, Affinity) for 60 min at 25 °C without light. Nuclei of cells were labeled with 4′,6-diamidino-2-phenylindole (DAPI).