Nucleofector kit 5

Short interfering RNAs (siRNAs) were obtained from Dharmacon (Lafayette, CO, USA). THP-1 cells were transfected with human Nrf2 siRNA (L-003755-00-0005) or nontargeting control siRNA (D-001810-10-05), each at a final concentration of 100 nM, using Nucleofector Kit V (Amaxa Biosystems, Cologne, Germany), according to the manufacturer's protocol with slight modifications. Following transfection, the cells were incubated with PMA for 72 hr, collected, lysed in 100 μl lysis buffer (10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 10 mM NaF, 2 mM Na₃VO₄, 1 mM PMSF, and 1% Triton X-100 with 1/20 v/v Complete), and sonicated for 10 s with an ultrasonic disruptor (Sonifier model 250, Branson Ultrasonics, Danbury, CT, USA). The resulting lysates were subjected to immunoblotting analysis. Nrf2 expression was measured by western blotting as above, with β-actin as the loading control. Detected bands were scanned, and intensities of chemiluminescence signals were quantified by ImageJ software. PMA-differentiated siRNA-transfected cells were further polarized into M1 macrophages in the presence or absence of propofol (50 μM), and IL-6, IL-1β, and TNF-α concentrations in supernatants were measured by ELISA.

Jurkat T cells were transiently transfected with cDNAs encoding GFP-tagged transgelin2 by Nucleofector Kit V (Amaxa) according to manufacturer’s instructions and then selected with 1 mg/mL Geneticin. Jurkat T cells (TIB-152, American Type Culture Collection) transfected with GFP-tagged transgelin2 were grown in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C. Jurkat T cells expressing GFP-tagged transgelin2 were grown to confluence and then subjected to serum deprivation for 18 h. These cells were subjected to each of the following treatment conditions. No-activation condition samples were untreated or treated with 100 nM calyculin A (LC Laboratories), or 1 μM okadaic acid for 30 min prior to cell harvest. PKA activation condition was induced by 100 nM forskolin and PKC activation was induced by 200 nM phorbol 12-myristate 13-acetate (PMA) for 30 min. Two protein kinase activation condition samples were then treated with 100 nM calyculin A for 30 min prior to cell harvest.