Ne per r nuclear and cytoplasmic extraction kit

A total of 10⁷ cells were rinsed with PBS once and then pelleted. Fractionation of the nuclear and cytoplasmic samples was performed using an NE-PER(R) nuclear and cytoplasmic extraction kit (Thermo Fisher). Total RNA in the nuclear and cytoplasmic fractions was extracted by Trizol. The nuclei–cytoplasm ratio was determined by the mRNA levels of targets in the nuclear and cytoplasmic fractions, which were normalized to the levels of nuclear MALAT1 RNA and cytoplasmic 7SL RNA, respectively [31 (link)].

Cells were washed twice with PBS and harvested in RIPA buffer. The nuclear proteins were extracted using NE-PER(R) Nuclear and Cytoplasmic Extraction kit (Thermo). Total cellular or nuclear extracts were separated with SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane. After blocking with 5% nonfat milk in Tris-buffered saline for 1 h at room temperature, the membrane was incubated with primary antibodies against AP-1 (Santa Cruz Biotechnology), RIG-I, TLR3, NF-κB, ERK, phospho-ERK, p38MAPK, and phospho-38MAPK (Cell Signaling Technology). After washing with Tris-buffered saline containing 0.5% (w/v) Tween [18 (link)], the membrane was incubated with an appropriate peroxidase-conjugated secondary antibody (Boster). Each blot was developed using SuperSignal West Pico and SuperSignal West Femto (Thermo). The images were captured using the MF-Chemi Bio Imaging System (DNR). To correct the results for potential variations in sample load, each blot was stripped and reprobed with anti-β-tubulin or anti-GAPDH (Santa Cruz Biotechnology). The fold changes of protein bands were analyzed with Image J software.