4 to 12 gradient gel

MCC-T hybridoma cells (50 × 10⁶) were stimulated as described earlier with anti-CD3 and anti-CD28 mAbs for 2 min at 37°C with gentle shaking, and resuspended in ice-cold buffer (0.25 M sucrose, 1 mM EGTA, 3 mM imidazole). Cells were disrupted by sonication and then homogenized with a 27-gauge needle. Nuclei were pelleted by centrifugation at 800g for 5 min, and supernatants were centrifuged at 1450g for 10 min at 4°C to obtain the membrane fraction (P1). Centrifugation at 17,000g for 10 min at 4°C was performed to obtain the organelle fraction (containing mitochondria, lysosomes, endosomes, and peroxisomes), which we refer to as the P2 fraction. A final centrifugation at 100,000g for 1 hour at 4°C was performed to separate the ER and Golgi (P3) from the cytosolic fraction (SN3). Fractions were resolved on a 4 to 12% gradient gel (Life Technologies) and were analyzed by Western blotting with anti–α1 sodium-potassium adenosine triphosphatase (ab7671, Abcam) or anti-p38 (#9212, Cell Signaling Technology) antibodies.