Protein IP Assay for Anti-EJ Detection

We established a protein IP assay to detect anti-EJ in patients with myositis. Human HEK293 cells (ATCC CRL-1573) were grown in DMEM supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) in a humidified atmosphere of 5% CO₂ at 37°C. Transient transfections were carried out using Megatran 2.0 (TT200003, Origene, USA) with pENTER-flag- GlyRS (glycyl tRNA synthetase) plasmid (CH810182, Vigene Biosciences, USA). After 48 hrs, cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) for western blot or protein immunoprecipitation. IP was performed according to protocols with minor modifications [16 (link), 17 (link)]. Immunoprecipitated antigens were solubilized in 1× SDS-PAGE loading buffer and separated by 10% SDS-PAGE and transferred to PVDF membrane. The membrane was then detected with anti-flag antibodies (1:1000, F1804, Sigma). The secondary antibody used was HRP-conjugated anti-mouse IgG (1:10000, GAM007, Multi Science).