Custom peptide and antibody production and testing
Pairs of phosphorylated and nonphosphorylated peptides were synthesized corresponding to FUS residues 22–36 (GQGYSQQSSQPYGQQ; Ser-26 underlined) and residues 26–40 (SQQSSQPYGQQSYSG; Ser-30 underlined). The phosphorylated peptides were used as immunogens for antibody production in rabbits. Following immunological challenge with each phosphopeptide, antibodies specific to the phosphorylated peptides were purified: anti-FUS(pSer26) and anti-FUS(pSer30). Nonspecific antibodies that recognized both PAN were also isolated. Peptide synthesis and antibody production were performed by Genscript (Piscataway, NJ).
Phosphorylated and nonphosphorylated peptides were blotted onto nitrocellulose and probed with anti-FUS(pSer26) and anti-FUS(pSer30) antibodies following normal Western blotting protocols. Knockdown of FUS from H4 cells using Lipofectamine RNAiMAX (ThermoFisher 13778030) and Silencer Select siRNA specific to FUS (ThermoFisher 4392420 and 4390843) was done following manufacturer’s protocol to confirm antibody specificity in cells.