Clone vin 11 5

Western blot was performed on protein extracts from muscles homogenized in tissue buffer and processed as described for MIP-2 ELISA assay. Total protein extracts of 30 μg were loaded for each sample. Images were acquired using Chemidoc ImageLab software (Bio-Rad). The following antibodies and dilutions were used: rabbit anti-Nfix (1:5000, Geneka Biotechnology), mouse anti-Vinculin (1:2500, Clone VIN-11-5, Sigma-Aldrich), and IgG-HRP secondary antibodies (1:10,000, Bio-Rad).

Cell lysates were obtained in RIPA buffer containing 1 mM PIC (Protease Inhibitor Cocktail, SIGMA), 10 mM NaF, 1 mM Na₃VO₄ and 0.5 mM DTT. 10–30 µg cell lysate was subjected to SDS-PAGE and transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore). For folate receptor beta (FOLR2), cell lysates were subjected to SDS-PAGE under non-reduced conditions. Protein detection was carried out using rabbit antibodies against pp38 and pERK (clones D3F9 and D13.14.4E, Cell Signaling, 1/1000), MAFB (HPA005653, Santa Cruz, 1/1000), pGSK3β (clone D85E12, Cell Signaling, 1/1000) and mouse monoclonal antibody against human CD163 (clone EDHu-1, Bio-Rad, 1/1000), pSTAT5 (clone 8-5-2, Millipore, 1/1000), FOLR2 (FRβ, kindly provided by Dr. Takami Matsuyama [32 (link)], dilution 1/800). Protein loading was normalized using an antibody against GAPDH (6C5, Santa Cruz Biotechnology, 1/2000) or against human vinculin (clone VIN-11-5, Sigma-Aldrich, 1/3000).