Spleen Tissue Western Blot Protein Extraction

Frozen spleen tissue was ground with mortar and pestle on dry ice, resuspended in cold RIPA buffer (Sigma-Aldrich, St Louis, MO, USA) containing a protease inhibitor and phosphatase inhibitor cocktail and then homogenized. The protein supernatant was collected and the protein concentration was determined using a bicinchoninic acid protein assay kit (BCA assay, Pierce, Rockford, IL, USA). The proteins were diluted in NuPAGE LDS Sample Buffer (4×) and denatured at 95 °C for 10 min. Following this, 50 µg of protein (in total 30 µL volume) was loaded per lane in a 4–12% Bis-Tris 18-well Criterion XT precast polyacrylamide gel (BioRad, Hercules, CA, USA). The gel was run at 70 V on the Criterion Midi format Cell™ (BioRad), then transferred using Trans-blot Turbo transfer system onto PVDF membrane. Blocking and incubation of membranes with primary and secondary antibodies were carried out using the Starting Block (ThermoFisher). Blots were washed using 0.05% TBST and TBS. The following antibodies were used: β-Actin (8457), JNK (9252), p-JNK (4668), AKT (9272), and pAKT (9271) from Cell Signaling. β-actin was used as the protein loading control. Densitometric analyses of protein bands were performed using Image Lab 5.2.1 Software from BioRad [33 (link)].