Western Blot Analysis of Protein Samples

Splenocytes were lysed on ice for 15 min with ice-cold RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS] supplemented with protease inhibitors (1 mM PMSF, 5 μg/ml aprotinin, and 5 μg/ml leupeptin) and phosphatase inhibitors (1 mM Na₃VO₄ and 1 mM NaF) and then centrifuged for 5 min at 12,000 rpm at 4°C. The protein concentrations were measured in a BCA protein assay (Pierce, Rockford, IL, USA) using BSA as a standard. Proteins (20 μg/lane) were separated by SDS-PAGE and transferred to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated with 5% skim milk for 1 h at room temperature and then washed with Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBS-T). The membranes were incubated overnight at 4°C with specific primary antibodies. After washing with TBS-T, the membranes were incubated for 1 h at room temperature with 5% skim milk containing peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat IgG. Signals were visualized using EZ-Western Lumi Femto (DoGenBio, Seoul, Korea) and quantified on a LAS-4000 (GE Healthcare Life Sciences, Marlborough, MA, USA). The intensity of the protein bands on each blot was quantified by densitometric analysis using ImageJ software (Bethesda, MD, USA) (28 (link), 29 (link)).