Cytoplasmic and Nuclear Protein Extraction

Cells were harvested with trypsin-EDTA and then centrifuged and resuspended in cold buffer A (10 mM HEPES pH 7.9, 10 mM KCl 0.1 mM EDTA, 0.1 mM EGTA 1 mM DTT, 1x protease inhibitor cocktail). Then after swelling on ice for 15 min, 10% solution of NP-40 was added and the tube was vigorously vortexed for 10 s. The cells lysate was centrifuged at 12,000 g for 1 min, and the supernatant was transferred as cytoplasmic extract. The nuclear pellet was resuspended in RIPA buffer. The conventional cytosolic protein was separated and transferred to PVDF membranes, subsequently examined by immunoblotting as previously described [30 (link)]. Primary antibodies against MMP1 (#54376; CST), Phospho-STAT3 Tyr705 (#9145; CST), STAT3 (#12640; CST), GAPDH (HRP-60004; Proteintech, China), Lamin A/C (10298-1-AP; Proteintech, China), Alpha Tubulin (HRP-66031; Proteintech, China) in 3% bovine serum albumin and secondary antibody (Mouse BA1050; Rabbit BA1054; Boster, China) were used. Protein bands were visualized using super enhanced chemiluminescence prime (S6008L; Bioscience, China).