Subcellular protein fractionation kit

The accumulation of DNA damage response proteins to chromatin following IR-induced DNA damage was examined using the Subcellular Protein Fractionation Kit (Thermo Fisher) as previously described (15 (link)). The cells were mock-treated or irradiated with 10 Gy, allowed to recover for 10 min, harvested after trypsinization, and then processed with the Thermo Fisher Subcellular Protein Fractionation Kit according to the manufacturer's instructions. The protein concentration of each sample was measured using a Pierce BCA Protein Assay kit (Thermo Fisher). 30 μg of the cytoplasmic fraction and 15 μg of the soluble nuclear extract or chromatin fraction were resolved via SDS-PAGE, and then transferred to a PVDF membrane for immunoblotting using the protocol outlined below.

LSMC were transfected with 50 nM pre-miR-200c or preNC for 48 hrs. Cell membrane, cytoplasmic, and nuclear proteins were fractionated following the protocol of the subcellular protein fractionation kit (Thermal Scientific, Waltham, MA).

1 x 10⁶ HeLa cells were infected at a MOI of 5. Cells and supernant were collected at 4 hpi and were lysed on ice for 5 min in RIPA lysis buffer supplemented with 1x Halt protease and phosphatase inhibitor cocktail. Cells were further processed for subcellular fractionation using a Subcellular Protein Fractionation Kit (ThermoFisher). Samples were mixed with Laemmli sample buffer (Bio-Rad), incubated at 95°C for 5 minutes, separated by SDS-PAGE, and transferred to PVDF membranes. Immunoblotting was performed using rabbit polyclonal anti-mCherry and rabbit monoclonal anti-EGFR (Abcam) followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signalling).

Western blotting was performed as previously described 16 (link). To estimate the distribution and cellular localization of specific proteins, the subcellular protein fractionation kit (#78833; Thermo Scientific, Waltham, MA, USA) was used to extract proteins from nuclear and cytoplasmic fractions, and the assay was conducted according to the manufacturer's instruction.

To aid in visualizing alterations in claudin-5 localization, whole cell lysates were fractionated using the Sub-cellular Protein Fractionation Kit (Thermo Fisher) according to the manufacturer’s instructions. Additionally, cells were dissociated using 0.25% trypsin-EDTA (Gibco Thermo Fisher Scientific) and pooled in 2 mL microcentrifuge tubes (Eppendorf, Hamburg, Germany). Next, they were washed with PBS and centrifuged at 500× g for 3 min to obtain the cell pellet. The reagents were added, followed by incubation and centrifugation steps to acquire the cytoplasmic and membrane fractions.