Total RNA was isolated from HEK-293 cell line and was dotted to NC membrane at different amount as indicated above the dots. The membrane was blocked with BSA and blotted with m6A antibody 68055-1-Ig at 1:2000 followed by incubation of HRP-goat anti-mouse secondary antibody. Signal was developed by ECL substrate. A parallel dot blot was performed using unrelated antibody with the same isotype (UCP2 antibody 66700-1-Ig) at the same dose.

Indirect ELISA and competitive ELISA results show that this antibody is specific to m6A. Indirect ELISA was performed by coating BSA conjugated m6A at 20ng/well followed by blocking with 1% BSA. Serial diluted primary antibody was added to the plates and incubated at 37℃. HRP-goat anti-mouse was used for detection. Competitive ELISA was performed similarly except that different concentration of m6A or its structure analogue compounds are mixed in primary antibody (fixed at 0.05μg/mL).

Immunohistochemical analysis of paraffin-embedded mouse testis tissue slide using 68055-1-Ig (m6A antibody) at dilution of 1:4000 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).

HEK-293 cells were lysised and immunoprecipitated with Protein A-m6A antibody and Protein A-mouse IgG3 control antibody respectively in the presence of RNAase inhibotor cocktail. The immunoprecipitated complex was washed diggested by RNAse A followed by western blot with YTHDF1(m6A reader) antibody 17479-1-AP (1:2000). (Lysate: 3.6mg per IP; IP: 15µg antibody and 50µL beads, 4 hours at 4℃; Diggestion: 50µg/mL * 80µL RNAse A for 1 hour at 37℃; Loading: 20% of elution; Input: 10µg.)

HEK-293 cells were lysised and immunoprecipitated with Protein A-m6A antibody and Protein A-mouse IgG3 control antibody respectively in the presence of RNAase inhibotor cocktail. The immunoprecipitated complex was washed diggested by RNAse A followed by western blot with IGF2BP3 (m6A reader) antibody 66526-1-Ig (1:2000). (Lysate: 4.0 mg per IP; IP: 30µg antibody and 50µL beads, 4 hours at 4℃; Diggestion: 50µg/mL * 80µL RNAse A for 1 hour at 37℃; Loading: 20% of elution; Input: 10µg.)