Western blot analysis

BV2 cells were plated at 1.5 × 10⁶ cells/well in 6‐well plates coated with r4‐1BB Fc or human IgG for 30 min. The r4‐1BB Fc‐treated BV2 cells were rinsed with PBS, suspended by scraping in lysis buffer (10 mm Tris/HCl, 10 mm NaCl, 0.1 mm EDTA, 50 mm NaF, 10 mm Na₄P₂O₇, 1 mm MgCl₂, 0.5% deoxycholate, 1% IGEPAL, protease inhibitors, phosphatase inhibitor cocktail), and centrifuged at 800 g for 5 min. Samples containing 15–30 μg of total protein were subjected to western blot analysis using polyclonal antibodies to IκB‐α (inhibitor of nuclear factor‐κB alpha; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p‐STAT3 (phospho‐signal transducer and activator of transcription 3; Cell Signaling, Danvers, MA, USA) and β‐actin (Sigma, St. Louis, MO, USA), p‐ERK (p‐extracellular signal‐regulated kinases), total ERK, p‐JNK (c‐jun amino‐terminal kinase), and total JNK (Cell Signaling).