Journal: Drug Design, Development and Therapy

Article Title: Immunomodulatory effects of 17- O -acetylacuminolide in RAW264.7 cells and HUVECs: involvement of MAPK and NF-κB pathways

doi: 10.2147/DDDT.S68659

Figure Lengend Snippet: Effects of different doses of AA pretreatment on LPS-induced MAPKs phosphorylation. Notes: RAW264.7 cells were grown to confluence and were starved in medium containing 2% FBS for 18 hours. The cells were then either left UT in medium or were pretreated with indicated concentrations of AA or equivalent dimethyl sulfoxide concentrations or the indicated MAPK inhibitors for 1 hour. The cells were then stimulated with 1 μg/mL LPS for 15 minutes or were left unstimulated. Proteins were extracted and detected by Western blotting, and images are representative of at least three independent experiments. When compared to the LPS-stimulated cells (0 μg/mL AA) and UT cells (0); AA-pretreated cells decreased all MAPKs phosphorylation at all concentrations for P-ERK ( A ) and at concentrations ranging from 1.25–5 μg/ml for P-JNK and P-p38 ( B and C , respectively) in the presence of LPS stimulation for 15 minutes. On the other hand, pretreatment with 10 μg/mL of AA increased P-JNK phosphorylation, ( B ) but not significantly for P-p38 ( C ). Abbreviations: AA, 17- O -acetylacuminolide; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; JNK, c-jun N terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; P-ERK, phosphorylated ERK; P-JNK, phosphorylated JNK, P-p38, phosphorylated p38; UT, untreated.

Article Snippet: The Mitogen-activated protein kinase kinase (MEK), MAPK, and p38 inhibitors U0126 (with a purity of 95%), PD98059 (>95%), SB203580 (98%), ICAM-1, VCAM-1, and E-selectin human enzyme-linked immunosorbent assay (ELISA) kits were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Phospho-proteomics, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Immunomodulatory effects of 17- O -acetylacuminolide in RAW264.7 cells and HUVECs: involvement of MAPK and NF-κB pathways

doi: 10.2147/DDDT.S68659

Figure Lengend Snippet: Quantified effects of different AA concentrations on LPS-induced MAPKs phosphorylation in RAW264.7 cells. Notes: When compared to the LPS-stimulated cells (dotted line set at a ratio of 1 in all three inset figures), UT cells, and AA-pretreated cells significantly decreased all MAPKs phosphorylation at all concentrations for P-ERK ( A ) and at concentrations ranging from 1.25–5 μg/mL for P-JNK and P-p38 ( B and C ) in the presence of LPS stimulation for 15 minutes. On the other hand, pretreatment with 10 μg/mL of AA significantly increased P-JNK phosphorylation, ( B ) but not significantly for P-p38 ( C ). Data are the average of two to three independent experiments and were analyzed with one-way ANOVA and Tukey’s post hoc test. * P <0.05; ** P <0.01; *** P <0.001. Abbreviations: AA, 17- O -acetylacuminolide; ANOVA, analysis of variance; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; P-ERK, phosphorylated extracellular signal-regulated kinase; P-JNK, phosphorylated JNK, P-p38, phosphorylated p38; UT, untreated.

Article Snippet: The Mitogen-activated protein kinase kinase (MEK), MAPK, and p38 inhibitors U0126 (with a purity of 95%), PD98059 (>95%), SB203580 (98%), ICAM-1, VCAM-1, and E-selectin human enzyme-linked immunosorbent assay (ELISA) kits were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Phospho-proteomics

Journal: Drug Design, Development and Therapy

Article Title: Immunomodulatory effects of 17- O -acetylacuminolide in RAW264.7 cells and HUVECs: involvement of MAPK and NF-κB pathways

doi: 10.2147/DDDT.S68659

Figure Lengend Snippet: Effects of AA alone on MAPKs phosphorylation. Notes: RAW264.7 cells were grown to confluence and were starved in medium containing 2% FBS for 18 hours. The cells were then either left UT in medium or were treated with 5 μg/mL and 10 μg/mL of AA for 1 hour. Cell lysates were separated on SDS-PAGE, transferred, and blotted against the specified antibodies. ( A ) Representative experiment of the effects of AA on MAPKs. ( B ) Quantified effects of AA on MAPK phosphorylation. The phosphorylation ratio of every MAPK is compared to the UT cells (0 μg/mL). Data are the average of two independent experiments and were analyzed with one-way ANOVA and Tukey’s post hoc test. * P <0.05; ** P <0.01. Phosphorylation of JNK is significantly different when compared to untreated cells. # P <0.05, ## P <0.01. Abbreviations: AA, 17- O -acetylacuminolide; ANOVA, analysis of variance; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; JNK, c-jun N terminal kinase; MAPK, mitogen-activated protein kinase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; P-ERK, phosphorylated ERK; P-JNK, phosphorylated JNK, P-p38, phosphorylated p38; UT, untreated.

Article Snippet: The Mitogen-activated protein kinase kinase (MEK), MAPK, and p38 inhibitors U0126 (with a purity of 95%), PD98059 (>95%), SB203580 (98%), ICAM-1, VCAM-1, and E-selectin human enzyme-linked immunosorbent assay (ELISA) kits were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Phospho-proteomics, SDS Page, Polyacrylamide Gel Electrophoresis

Journal: FASEB BioAdvances

Article Title: RBM25 Regulates p38 MAPK Pathway Activation via Exon 16 Skipping of MAP4K4 in a Rat Model of Post‐Infarction Heart Failure

doi: 10.1096/fba.2025-00201

Figure Lengend Snippet: RBM25 regulates exon 16 skipping of MAP4K4. (A) PCR amplification of CSF1; (B) PCR amplification of MAP4K4‐a/b. The gel image shows the following groups: A: Sham, B: HF(heart failure model), C: OE‐NC(empty vector overexpression), D: OE‐RBM25(RBM25 overexpression), E: E‐RBM25 + SB203580(p38 inhibitor), F: sh‐NC (scramble shRNA), G: sh‐RBM25 (RBM25 knockdown), H: sh‐RBM25 + Gambogic Amide MAPK activation; (C) Structural Consequences of ΔEx16 Cartoon representations of AlphaFold3‐predicted structures: Wild‐type MAP4K4(Green); Exon 16‐deficient MAP4K4‐ΔEx16 (Pink).ΔEx16 induces conformational rearrangement in the kinase domain (circled region); (D) Protein–protein docking simulations via HDOCK. Predicted binding interfaces and quantified affinities between MAP3K1 (Purple) and wild‐type MAP4K4 (Green) versus MAP4K4‐ΔEx16 (Pink).

Article Snippet: SB203580 (5 mg/kg/day; MEC, RWJ 64809) or Gambogic Amide (2 mg/kg/day; Alomone Labs, G‐235) was injected intraperitoneally daily for 8 weeks.

Techniques: Amplification, Plasmid Preparation, Over Expression, shRNA, Knockdown, Activation Assay, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Differential Inflammatory-Response Kinetics of Human Keratinocytes upon Cytosolic RNA- and DNA-Fragment Induction

doi: 10.3390/ijms19030774

Figure Lengend Snippet: Activation of Nuclear Factor κB (NF-κB), Mitogen Activated Protein Kinase (MAPK) and Signal Transducers of Activator of Transcription (STAT) signal transduction pathways in HPV-KER cells upon poly(I:C) or poly(dA:dT) transfection assessed by NF-κB-luciferase reporter assay ( A ) and western blot analysis ( B – D ). ( A ) NF-κB luciferase reporter assay exhibited faster activation of NF-κB transcription factor upon poly(I:C) treatment than poly(dA:dT) treatment. Raw luminescence intensity values were normalized to the intrinsic control renilla activity, and compared to the 0 h untreated samples. Data are presented as mean of three independent experiments ± standard error; statistical significance was assessed by two-way repeated measurement ANOVA * p < 0.05, grey: poly(I:C) treatment compared to untreated 0 h samples, black: poly(dA:dT) treatment compared to untreated 0 h samples; ( B ) Increase in phosphorylated NF-κB inhibitor α (IκBα) after poly(I:C) or poly(dA:dT) treatment, peaking later after poly(dA:dT) treatment than after poly(I:C) treatment, arrow indicate the lane for phosphorylated IκBα; ( C ) Phosphorylation of ERK1/2 increases after poly(I:C) or poly(dA:dT) treatment, peaking later after poly(dA:dT) treatment than after poly(I:C) treatment, arrows indicate from top to bottom the lanes for phosphorylated ERK1 and ERK2. Phosphorylation of p38 and JNK was not observed upon poly(I:C) or poly(dA:dT) treatment; ( D ) Phosphorylation of both STAT-1 and STAT-3 occurs faster in poly(I:C) treated samples than in poly(dA:dT) treated samples. Western blot results are representative for at least three independent experiments. Actin was used as loading control.

Article Snippet: For inhibition studies, cells were incubated 1 h prior to poly(dA:dT)/poly(I:C) transfection with inhibitors for NF-κB (Bay 11-7085, 10 μM; MedChem Express, Monmouth Junction, NJ, USA), STAT-1 (Fludarabine, 10 μM; Sigma Aldrich), STAT-3 (Stattic, 5 μM; Sigma Aldrich), MEK1 (PD98059, 20 μM; Sigma Aldrich), JNK (SP600125, 10 μM; Tocris Bioscience, Bristol, UK) and p38 (SB203580, 10 μM; Tocris Bioscience).

Techniques: Activation Assay, Transduction, Transfection, Luciferase, Reporter Assay, Western Blot, Control, Activity Assay, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Differential Inflammatory-Response Kinetics of Human Keratinocytes upon Cytosolic RNA- and DNA-Fragment Induction

doi: 10.3390/ijms19030774

Figure Lengend Snippet: Inhibition of different signaling routes has divergent effects on the expression of the IL-6 (white bars) and TNF-α (grey bars) cytokines in keratinocytes. The effect of inhibition by NF-κB ( A ), p38 ( B ), c-Jun N-terminal kinase (JNK) ( C ), mitogen-activated protein kinase kinase 1 and2 (MEK1/2) ( D ), STAT-1 ( E ) and STAT-3 ( F ) on poly(I:C) (6 h after transfection) and poly(dA:dT) (12 h after transfection) induction of IL-6 (white bars) and TNF-α (grey bars) expression in HPV-KER cells. Fold change of mRNA expression values were determined by the ΔΔ C t method, normalized to 18S rRNA expression. As all inhibitors were dissolved in dimethyl sulfoxide (DMSO), the relative mRNA expression levels were compared to the expression levels in samples treated with DMSO + poly(I:C) or DMSO + poly(dA:dT), respectively. Poly(I:C) and poly(dA:dT) induction was in every case significant compared to the untreated control samples; no significant difference was observed between the cytokine-expression level of the samples treated with poly(I:C), poly(dA:dT), DMSO + poly(I:C) or DMSO + poly(dA:dT). Data are represented as the means of three independent experiments ± standard error; * p < 0.05; ** p < 0.01; *** p < 0.001 determined by Student’s t -test.

Article Snippet: For inhibition studies, cells were incubated 1 h prior to poly(dA:dT)/poly(I:C) transfection with inhibitors for NF-κB (Bay 11-7085, 10 μM; MedChem Express, Monmouth Junction, NJ, USA), STAT-1 (Fludarabine, 10 μM; Sigma Aldrich), STAT-3 (Stattic, 5 μM; Sigma Aldrich), MEK1 (PD98059, 20 μM; Sigma Aldrich), JNK (SP600125, 10 μM; Tocris Bioscience, Bristol, UK) and p38 (SB203580, 10 μM; Tocris Bioscience).

Techniques: Inhibition, Expressing, Transfection, Control

Journal: The Journal of biological chemistry

Article Title: Cell volume-dependent regulation of L-selectin shedding in neutrophils. A role for p38 mitogen-activated protein kinase.

doi: 10.1074/jbc.274.31.22072

Figure Lengend Snippet: FIG. 7. The role of p38 mitogen-activated protein kinase in the shedding of L-selectin induced by hypertonicity and other stim- uli. A, neutrophils were treated with either genistein (40 mM) or PP1 (10 mM) for 20 min and then challenged with hypertonicity (100 mM extra NaCl) for 10 min or 2 h. Cells were lysed, subjected to SDS-PAGE, and immunoblotted using polyclonal anti-phospho-p38. The same blot was then stripped and reprobed with an anti-p38 antibody. B, following the same treatment as in A and exposure to hypertonicity for 2 h, p38 was immunoprecipitated with a specific anti-p38 antibody. In vitro kinase assay was then performed using ATF-2 as a substrate, and the results were visualized with autoradiography. C, neutrophils were treated in the presence or absence of the p38 inhibitor SB203580 (20 mM) for 20 min. The medium was then supplemented with an extra 100 mM NaCl for 2 h (HT), LPS (1 mg/ml) for 1 h, FMLP (100 nM), or PMA (50 nM) both for 20 min. L-selectin surface expression was measured using a mAb and flow cytometry. The data represent the means 6 S.E., n 5 6 separate studies. *, p , 0.001 versus control (CON); **, p , 0.05 versus control; ***, p , 0.01 versus control; Genist., genistein; HT, hypertonic medium.

Article Snippet: FMLP, PMA, urea, sucrose, lipopolysaccharide (Escherichia coli O111: B4), bovine serum albumin, diisopropylfluorophosphate, and propidium iodide were from Sigma, and Triton X-100 was from Caledon Lab. BCECF was obtained from Molecular Probes Inc.; genistein, erbstatin analog, SB203580, DEVD-fmk, and staurosporin were from Calbiochem; the proteinase inhibitor mixture was from PharMingen; and ATF-2 was from Santa Cruz Biotechnology.

Techniques: SDS Page, Immunoprecipitation, In Vitro, Kinase Assay, Autoradiography, Expressing, Flow Cytometry, Control

Journal: Experimental eye research

Article Title: Gremlin utilizes canonical and non-canonical TGFβ signaling to induce lysyl oxidase (LOX) genes in human trabecular meshwork cells.

doi: 10.1016/j.exer.2013.05.011

Figure Lengend Snippet: Fig. 8. p38 MAPK inhibitor SB203580 blocks gremlin-induction of several LOX proteins. Representative western immunoblots of p38 MAPK inhibitor SB203580 effects on gremlin induction of cell-associated (A) and secreted (B) LOX proteins. Western immunoblots of TM cells pretreated with 10 mM of SB203580 followed by treatment with 1 mg/ml of gremlin for 24 h. Untreated and inhibitor only treated cells served as negative controls. GAPDH was used as loading control for cell associated LOXs. Blots shown are representative data obtained in one TM strain. The experiment was performed independently two times in three different TM cell strains. (CeD) Densitometry analysis of western immunoblots for cell associated (C) and secreted (D) LOXs are plotted as bar graphs. Two-tailed student’s t test was used to compare gremlin- and gremlin þ SB203580-treated samples. **0.0001 < p < 0.01, ***p < 0.0001.

Article Snippet: SMAD-3 phosphorylation inhibitor SIS3 (10 mM, SigmaeAldrich, St. Louis, MO), JNK inhibitor SP600125 (10 mM, SigmaeAldrich, St. Louis, MO), and p38 MAPK inhibitor SB203580 (5 mM, Tocris Biosciences, Ellisville, MO) were used to examine effects of inhibition on canonical SMAD, JNK, and p38 signaling pathways, respectively (Sethi et al., 2011b).

Techniques: Western Blot, Control, Two Tailed Test

Journal: Experimental eye research

Article Title: Gremlin utilizes canonical and non-canonical TGFβ signaling to induce lysyl oxidase (LOX) genes in human trabecular meshwork cells.

doi: 10.1016/j.exer.2013.05.011

Figure Lengend Snippet: Fig. 9. Proposed mechanism of gremlin regulation of LOXs in TM. Gremlin blocks BMP signaling pathway, which is unable to inhibit the endogenous TGFb signaling in TM cells. The endogenously active TGFb pathway can activate Smad, P38 and JNK pathways to induce different LOX genes and proteins in TM cells.

Article Snippet: SMAD-3 phosphorylation inhibitor SIS3 (10 mM, SigmaeAldrich, St. Louis, MO), JNK inhibitor SP600125 (10 mM, SigmaeAldrich, St. Louis, MO), and p38 MAPK inhibitor SB203580 (5 mM, Tocris Biosciences, Ellisville, MO) were used to examine effects of inhibition on canonical SMAD, JNK, and p38 signaling pathways, respectively (Sethi et al., 2011b).

Techniques:

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Sensitization of colonic nociceptors by IL-13 is dependent on JAK and p38 MAPK activity.

doi: 10.1152/ajpgi.00280.2022

Figure Lengend Snippet: Figure 2. Activation of DRG sensory neurons by IL-13 is JAK1/2- and p38 MAPK-dependent. The proportion of IL-13-sensitive neurons was signifi- cantly reduced by pan-JAK inhibition with pyridone 6 (P6) (n = 5), JAK1/2 inhibition with ruxolitinib (Rux) (n = 5), and p38 MAPK inhibition with SB203580 (SB) (n = 5). DRG, dorsal root ganglion. P < 0.01.

Article Snippet: SB203580 (10 mM; DMSO; Tocris, Cat. No. 1402), pyridone 6 (1 mM; DMSO; Tocris, Cat. No. 6577), and ruxolitinib (1 mM; DMSO; Tocris, Cat. No. 7064) were purchased from Tocris and stock concentrations made up as described.

Techniques: Activation Assay, Inhibition

Journal: The Journal of biological chemistry

Article Title: p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens.

doi: 10.1074/jbc.275.4.2322

Figure Lengend Snippet: FIG. 1. Inhibition of tBHQ-induced p38 activation by SB203580. HepG2 (A) or Hepa1c1c7 (B) cells were incubated with SB203580 (5 mM), SB202474 (5 mM), or solvent (0.1% Me2SO) for 1 h prior to challenge with tBHQ (100 mM) for an additional 1 h. Cells were harvested, and the endogenous p38 kinase activity was determined by immunocomplex kinase assays using 5 mg of GST-ATF2-(1–96) fusion protein as substrate. The protein level of p38 was determined by West- ern blotting. The experiment was repeated three times.

Article Snippet: The specific p38 inhibitor, SB203580, and its negative analog, SB202474, were purchased from Calbiochem. tBHQ, BHA, and b-NF were purchased from Aldrich, and SUL was purchased from LKT Laboratories (St. Paul, MN).

Techniques: Inhibition, Activation Assay, Incubation, Solvent, Activity Assay

Journal: The Journal of biological chemistry

Article Title: p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens.

doi: 10.1074/jbc.275.4.2322

Figure Lengend Snippet: FIG. 2. Enhancement of tBHQ-induced QR activity by SB203580. Hepa1c1c7 cells were treated with SB203580 (5 mM), SB202474 (5 mM), or solvent (0.1% Me2SO) for 1 h and then stimulated with tBHQ (100 mM) for 24 h. Cells were harvested and assayed for QR activity by measuring the reduction of 2,6-dichloroindophenol as de- scribed under “Materials and Methods.” The data shown are means of four independent experiments 6 S.D.

Article Snippet: The specific p38 inhibitor, SB203580, and its negative analog, SB202474, were purchased from Calbiochem. tBHQ, BHA, and b-NF were purchased from Aldrich, and SUL was purchased from LKT Laboratories (St. Paul, MN).

Techniques: Activity Assay, Solvent

Journal: The Journal of biological chemistry

Article Title: p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens.

doi: 10.1074/jbc.275.4.2322

Figure Lengend Snippet: FIG. 3. SB203580 potentiates the induction of ARE-luciferase reporter gene by tBHQ. A, HepG2 cells were transiently transfected with 0.5 mg of pCH110-b-gal plasmid and 1.5 mg of ARE-TI-luciferase reporter construct or the construct without ARE enhancer (TI-Luc). After transfection, cells were incubated for 12 h in culture medium and then incubated with SB203580 (5 mM) or SB202474 (5 mM) for 1 h, prior to treatment with tBHQ (100 mM) for 24 h. Luciferase activity was determined and normalized against b-galactosidase activity. The amount of luciferase activity in the cells that were transfected with ARE-luciferase construct but treated with solvent alone was given an arbitrary value of 1. B, HepG2 cells were transfected with an ARE- luciferase reporter construct, and stably transfected cell clones were selected as described under “Materials and Methods.” C4, a randomly selected positive clone, was treated with the indicated concentrations of SB203580 or SB202474 for 1 h before the addition of tBHQ (100 mM). Luciferase activity was determined 24 h after treatment and normal- ized against protein concentration. The level of luciferase activity in untreated C4 cells was arbitrarily set to 1. The data shown are means of three independent experiments performed in duplicate.

Article Snippet: The specific p38 inhibitor, SB203580, and its negative analog, SB202474, were purchased from Calbiochem. tBHQ, BHA, and b-NF were purchased from Aldrich, and SUL was purchased from LKT Laboratories (St. Paul, MN).

Techniques: Luciferase, Transfection, Plasmid Preparation, Construct, Incubation, Activity Assay, Solvent, Stable Transfection, Clone Assay, Protein Concentration

Journal: The Journal of biological chemistry

Article Title: p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens.

doi: 10.1074/jbc.275.4.2322

Figure Lengend Snippet: FIG. 5. Effects of BHA, b-NF, and SUL on ARE-mediated gene expression and p38 activity. A, augmentation of BHA, b-NF, and SUL induction of ARE-mediated reporter gene activity by SB203580. Stably transfected C4 cells were pretreated with SB203580 (5 mM), SB202474 (5 mM), or solvent (0.1% Me2SO) for 1 h prior to the addition of BHA (100 mM), b-NF (5 mM), SUL (12.5 mM), or solvent. Luciferase activity was determined 24 h after drug treatment and normalized by protein concentration. The data, as expressed as -fold induction over control (solvent-treated cells), are means and S.D. values of four inde- pendent experiments. B, activation of p38 by BHA, b-NF, and SUL. C4 cells were untreated or pretreated with SB203580 (5 mM) before incu- bation with BHA (100 mM), b-NF (5 mM), or SUL (12.5 mM) for 1 h. The endogenous p38 activity was immunoprecipitated with a specific anti- body and assayed with GST-ATF2-(1–96) fusion protein as substrate. The protein level of p38 was determined by Western blotting. The experiment was repeated twice.

Article Snippet: The specific p38 inhibitor, SB203580, and its negative analog, SB202474, were purchased from Calbiochem. tBHQ, BHA, and b-NF were purchased from Aldrich, and SUL was purchased from LKT Laboratories (St. Paul, MN).

Techniques: Gene Expression, Activity Assay, Stable Transfection, Transfection, Solvent, Luciferase, Protein Concentration, Control, Activation Assay, Immunoprecipitation, Western Blot

Journal: The Journal of biological chemistry

Article Title: p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens.

doi: 10.1074/jbc.275.4.2322

Figure Lengend Snippet: FIG. 6. SB203580 induces an earlier kinetics of ARE-dependent gene activation than tBHQ. C4 cells were treated with SB203580 (5 mM) or tBHQ (100 mM) for different times. Luciferase activity was determined and normalized by protein concentration. The data ob- tained from three separate experiments were expressed as -fold induc- tion over control (solvent-treated cells).

Article Snippet: The specific p38 inhibitor, SB203580, and its negative analog, SB202474, were purchased from Calbiochem. tBHQ, BHA, and b-NF were purchased from Aldrich, and SUL was purchased from LKT Laboratories (St. Paul, MN).

Techniques: Activation Assay, Luciferase, Activity Assay, Protein Concentration, Control, Solvent