Keygen nuclear and cytoplasmic protein extraction kit

For fluorescent quantification of the total receptor expression, VPAC1-CHO and VPAC1-C37/A-CHO and CHO cells at logarithmic phases were collected, counted and lysised by ultrasonication fully, and the rough lysate were submitted to the fluorescent quantification on the multifunctional fluorescence detector Victor3 1420 (PE, USA) with exciting/emission light wavelengths of 460 ± 30 nm/535 ± 30 nm. And the expression of EYFP-tagged receptor was quantified using the formula: Unit cell fluorescence value = the light densities/ cells counts. The experiments were performed in parallel with at least five replicates and were repeated three times. For fluorescent quantification of the receptor transporting into the nuclear, the cell lysates of VPAC1-CHO and VPAC1-C37/A-CHO cells were submitted to the nuclear extraction using KeyGEN Nuclear and Cytoplasmic Protein Extraction Kit (KeyGEN, Shanghai, China), and the nuclear fraction were further lysis by ultrasonication then were submitted to the fluorescence quantification and protein concentration quantification using BCA protein assay kit (KeyGEN, Shanghai, China). The intranuclear EYFP-tagged receptor was calculated using the formula: The intranuclear receptor = the fluorescence densities in nucleus /protein concentration of nucleus.

GC cells (SGC7901, MGC803, MKN45 and MKN28) and the gastric epithelial cell line GES-1 are purchased from ATCC (Rockville, USA). Cells are cultured in Dulbecco's modified Eagles medium (DMEM)(Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS) (Invitrogen) supplemented with 1% penicillin-streptomycin (Invitrogen) at 37°C under 5% CO₂. Total proteins in cells are prepared using RIPA lysis buffer (Beyotime, Nanjing, China). The immunoblotting assay is performed with antibodies against TNFAIP6 (Proteintech Group, IL, USA), β-catenin (Proteintech), N-Cadherin (Proteintech), PTX3 (Proteintech), E-Cadherin (Proteintech), Lamin B1 (Proteintech), and GAPDH (Bioworld Technology) as previously described. Nuclear and cytoplasmic fractions are separated using a KeyGEN nuclear and cytoplasmic protein extraction kit (KeyGEN BioTECH, Nanjing, China). Secondary antigoat antibodies are purchased from Beyotime Biotechnology (Nanjing, China).

Total proteins were extracted using RIPA buffer (50 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) supplemental with protease inhibitors (Roche). KeyGEN Nuclear and Cytoplasmic Protein Extraction Kit (KGP150, KeyGEN BioTECH) was used to isolate nuclear proteins. Antibodies against MCM3 (ab4460, Abcam), p65 (ab16502, Abcam), p84 (ab487, Abcam), IKKβ (ab124957, Abcam), p-IKKβ (ab38515, Abcam), IκBα (ab32518, Abcam), p-IκBα (ab133462, Abcam), DNA PKcs (ab32566), DNA PKcs (phosphor S2056) (ab18192), CLEAVED PARP1 (ab32064) and GAPDH (G8795, Sigma).