Lture plates. They were treated with DCLF alone or in combination with TNF and/or IFN and also in the presence or absence of BAPTA/AM or 2-APB. For all studies involving BAPTA/AM, cells were pretreated with BAPTA/ AM for 4 h prior to the addition of DCLF and cytokines. For all studies involving 2-APB, cells were treated with 2-APB simultaneously with DCLF and cytokines. Cells were lysed and centrifuged after 24 h of exposure. 50 ll of lysate were added to blackwalled, 96-well plates and incubated with assay reaction buffer and fluorogenic substrate for 1 h. The plate was then read in a fluorescence plate reader at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Protein isolation. Cells (1.2 ?106 per well) were plated in 6-well tissue culture plates and allowed to adhere overnight. They were exposed to 250 lM DCLF and its vehicle alone or in combination with TNF and/or IFN for 18 h. For some experiments, cells treated with DCLF/cytokine combinations were also incubated in the presence of BAPTA/AM, 2-APB, or the JNK inhibitor,SP600125. SP600125 was prepared in DMSO and 0.1 DMSO was used as the vehicle control in all experiments involving treatment with SP600125. Cells were rinsed with cold PBS followed by addition of 150 ml of radioimmunoprecipitation assay buffer containing HALT protease and phosphatase inhibitor cocktails (Thermo Scientific, Rockford, Illinois). Cells were scraped, collected, placed in microcentrifuge tubes, and incubated on ice for 10 min. During the 10-min incubation, the tubes were vortexed intermittently. Lysates were centrifuged for 25 min at 20 000 ?g. The supernatant fluids containing whole cell protein were collected and stored at ?0 C until use. Protein concentrations were quantified using the order Baicalein 6-methyl ether bicinchoninic acid assay (Thermo Scientific). Western analysis. For detection of phosphorylated JNK (pJNK), phosphorylated ERK (pERK), phosphorylated PERK (pPERK), and phosphorylated STAT-1 (pSTAT-1) in whole cell lysates, 25 lg protein were loaded onto precast NuPAGE 12 Bis-Tris gels (Life Technologies), and subjected to electrophoresis. Proteins were transferred onto polyvinylidene fluoride membranes (Millipore).MAIURI ET AL.|FIG. 6. Ca�� contributes to DCLF-mediated ERK Quinagolide (hydrochloride)MedChemExpress CV205-502 hydrochloride activation. HepG2 cells were treated with VEH (0.1 DMSO), (A) BAPTA/AM (10 lM, 4 h before addition of DCLF/cytokines) or (B) 2-APB (100 lM, simultaneous addition with DCLF/cytokines) and treated with sterile water (Control) or DCLF (250 mM) alone or in combination with TNF (10 ng/ml) and/or IFN (10 ng/ml). Proteins were collected 18 h after drug treatment. pERK and a-tubulin were detected via western analysis. a, significantly different from Control group within a cytokine treatment. b, significantly different from BAPTA/AM (A) or 2-APB (B) within a cytokine treatment group. c, significantly different from DCLF within a cytokine treatment. Western analysis of proteins from cells treated with and without BAPTA/AM or 2-APB was performed simultaneously. Data are represented as mean 6 SEM of at least 3 experiments. Abbreviations: VEH, vehicle; DCLF, diclofenac; pERK, phosphorylated extracellular signal-regulated kinase; BAPTA/AM, acetoxymethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid; APB, aminophenoxydiphenyl borate.Membranes were blocked for 1 h with 5 bovine serum albumin (BSA) reconstituted in 1 tris-buffered saline (TBS) containing 0.1 tween-20 (TBSt). They were then probed with antibodies directed agains.Lture plates. They were treated with DCLF alone or in combination with TNF and/or IFN and also in the presence or absence of BAPTA/AM or 2-APB. For all studies involving BAPTA/AM, cells were pretreated with BAPTA/ AM for 4 h prior to the addition of DCLF and cytokines. For all studies involving 2-APB, cells were treated with 2-APB simultaneously with DCLF and cytokines. Cells were lysed and centrifuged after 24 h of exposure. 50 ll of lysate were added to blackwalled, 96-well plates and incubated with assay reaction buffer and fluorogenic substrate for 1 h. The plate was then read in a fluorescence plate reader at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Protein isolation. Cells (1.2 ?106 per well) were plated in 6-well tissue culture plates and allowed to adhere overnight. They were exposed to 250 lM DCLF and its vehicle alone or in combination with TNF and/or IFN for 18 h. For some experiments, cells treated with DCLF/cytokine combinations were also incubated in the presence of BAPTA/AM, 2-APB, or the JNK inhibitor,SP600125. SP600125 was prepared in DMSO and 0.1 DMSO was used as the vehicle control in all experiments involving treatment with SP600125. Cells were rinsed with cold PBS followed by addition of 150 ml of radioimmunoprecipitation assay buffer containing HALT protease and phosphatase inhibitor cocktails (Thermo Scientific, Rockford, Illinois). Cells were scraped, collected, placed in microcentrifuge tubes, and incubated on ice for 10 min. During the 10-min incubation, the tubes were vortexed intermittently. Lysates were centrifuged for 25 min at 20 000 ?g. The supernatant fluids containing whole cell protein were collected and stored at ?0 C until use. Protein concentrations were quantified using the bicinchoninic acid assay (Thermo Scientific). Western analysis. For detection of phosphorylated JNK (pJNK), phosphorylated ERK (pERK), phosphorylated PERK (pPERK), and phosphorylated STAT-1 (pSTAT-1) in whole cell lysates, 25 lg protein were loaded onto precast NuPAGE 12 Bis-Tris gels (Life Technologies), and subjected to electrophoresis. Proteins were transferred onto polyvinylidene fluoride membranes (Millipore).MAIURI ET AL.|FIG. 6. Ca�� contributes to DCLF-mediated ERK activation. HepG2 cells were treated with VEH (0.1 DMSO), (A) BAPTA/AM (10 lM, 4 h before addition of DCLF/cytokines) or (B) 2-APB (100 lM, simultaneous addition with DCLF/cytokines) and treated with sterile water (Control) or DCLF (250 mM) alone or in combination with TNF (10 ng/ml) and/or IFN (10 ng/ml). Proteins were collected 18 h after drug treatment. pERK and a-tubulin were detected via western analysis. a, significantly different from Control group within a cytokine treatment. b, significantly different from BAPTA/AM (A) or 2-APB (B) within a cytokine treatment group. c, significantly different from DCLF within a cytokine treatment. Western analysis of proteins from cells treated with and without BAPTA/AM or 2-APB was performed simultaneously. Data are represented as mean 6 SEM of at least 3 experiments. Abbreviations: VEH, vehicle; DCLF, diclofenac; pERK, phosphorylated extracellular signal-regulated kinase; BAPTA/AM, acetoxymethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid; APB, aminophenoxydiphenyl borate.Membranes were blocked for 1 h with 5 bovine serum albumin (BSA) reconstituted in 1 tris-buffered saline (TBS) containing 0.1 tween-20 (TBSt). They were then probed with antibodies directed agains.
