Minute cytoplasmic nuclear extraction kit

HSV-2–infected or romidepsin-treated cells were fractionated with Minute Cytoplasmic & Nuclear Extraction Kits (SC-003, Invent Biotechnologies Inc.), and at least 0.5 μg of nuclear extract was used to determine HDAC activity (Epigenase HDAC Activity/Inhibition Direct Assay Kit, P-4034-96, EpigenTek). Absorbance was measured using a SpectraMax M5e microplate reader and SoftMax Pro 7.1 GxP software (Molecular Devices).

Cytoplasmic and nuclear fractions were prepared from 5 × 10⁷ cells by using Minute™ Cytoplasmic & Nuclear Extraction Kits (Invent Biotechnologies, USA). Briefly, cells were washed with ice-cold PBS and lysed for 30 min on ice with cytoplasmic lysis buffer. The lysates were centrifuged at 14000 rpm for 5 min at 4 °C, and the supernatants (cytoplasmic fractionation) were collected in a fresh tube kept on ice. The pellet was resuspended with nuclear lysis buffer. The homogenate was incubated on ice for 30 min on ice and centrifuged at 14000 rpm for 5 min at 4 °C. Supernatant was collected as the nuclear fraction. The cytoplasmic and nuclear fractions were then analyzed using western blot.

The total protein was extracted from cells lysed with sodium dodecyl sulfate (SDS, Beyotime Biotechnology, Shanghai, China) lysate containing 1% phenylmethanesulfonyl fluoride (Beyotime Biotechnology) and 1% phosphatase inhibitor. The cytoplasmic and nuclear proteins were isolated with the Minute™ Cytoplasmic & Nuclear Extraction Kit (#SC-003; Invent Biotechnologies, Inc, Minnesota, USA) according to the manufacturer's instructions. The protein concentration was measured with the BCA protein assay kit (Beyotime Biotechnology). Protein samples were run on SDS-polyacrylamide gel electrophoresis (SDS- PAGE) and transferred to PVDF membranes. After blocking with 5% nonfat milk for 1 h at room temperature, the membrane was incubated with the primary antibody at 4 ℃ overnight. The following antibodies were used in the study: anti-CDK14 antibody (1:1000 dilution, Santa Cruz Biotechnology, Inc, Dallas, Texas USA), anti-MDR1 (1:5000 dilution, P-gp, Cell Signaling Technology, Inc., Boston, MA, USA), and anti-β-actin (1:5000 dilution, Proteintech, Wuhan, China). The secondary antibodies of the anti-mouse IgG or anti-rabbit IgG were used at RT for 1 h. The protein bands were photographed by the chemiluminescence imaging system (Tanon Science & Technology, Shanghai, China).

Nuclear and cytoplasmic proteins of cardiac tissues were extracted through MINUTE Cytoplasmic & Nuclear Extraction Kit (Invent Biotechnologies) following the manufacturer’s instructions. Antibodies against PPP3CA (Proteintech; catalog 13422-1-AP; 1:2000), NFATc3 (Santa Cruz Biotechnology; catalog sc-8405; 1:2000), HSP90 (EnoGene Biotech Co., Ltd.; catalog E1A0013B; 1:500), and PCNA (Proteintech; catalog 10205-2-AP; 1:1000) were used as primary antibodies, and a horseradish peroxidase–conjugated goat anti-rabbit or anti-mouse antibody (TransGen Biotech Co., LTD.; catalog HS101-01 or HS201-01; 1:10,000) was used as a secondary antibody.