Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: NO governs the activities of CYP2D6 and 3A4 in cells by modulating their heme content in a concentration-dependent bimodal fashion. GlyA-CHO cells underwent transfection to express FLAG- and MYC-tagged CYP3A4 or CYP2D6 in media enriched with 14 C Gly for 48 h, after which protein expression was ceased by Chx addition, and NOC18 was added at the designated concentrations. The cultures were maintained for 6 h before harvesting, followed by supernatant CYP activity assay and MYC Ab pulldown of the CYP to determine heme contents. A and B, CYP2D6 and CYP3A4 activities. C and D, CYP2D6 and CYP3A4 14 C heme counts. Data are the mean ± SD; n = 3 independent experiments. ∗∗∗ p < 0.001, one-way ANOVA. Ab, antibody; Chx, cycloheximide; CYP, cytochrome P450; 14 C-Gly, 14 C-glycine; GlyA-CHO, glycine auxotroph Chinese hamster ovary cell; NO, nitric oxide; NOC18, 2,2′-(hydroxynitrosohydrazino)bis-ethanolamine; UT, untransfected cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Concentration Assay, Transfection, Expressing, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: NO governs the activities of CYP2D6 and 3A4 within HepG2 cells. HepG2 cells underwent transfection to express FLAG- and MYC-tagged CYP2D6 ( A ) or were induced by rifampicin to express CYP3A4 ( B ) in media enriched with 14 C Gly, after which protein expression was ceased by Chx addition and NOC18 was added to give the designated concentrations. After 6 h, cells were harvested, and the supernatants were subjected to a CYP activity assay. Data are the mean ± SD; n = 3 independent experiments. ∗∗∗ p < 0.001, ∗∗ p < 0.01, one-way ANOVA. Chx, cycloheximide; CYP, cytochrome P450; HepG2, hepatocellular carcinoma G2 cell line; NO, nitric oxide; NOC18, 2,2′-(hydroxynitrosohydrazino)bis-ethanolamine; UT, untransfected cell samples; UI, uninduced cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Transfection, Expressing, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: NO generated by immune-stimulated macrophage cells modulates the heme level and activity of CYP3A4 expressed in cocultured GlyA-CHO cells. A, outline of the experiment; RAW264.7 cells that had been activated or not for NO synthesis by culture with bacterial LPS were added in varying numbers to monolayers of GlyA-CHO cells expressing CYP3A4 incorporated with 14 C heme by previous culture with 14 C-Gly. Following 6 h of coulture, the nitrite concentration in the culture fluid was quantified ( B ) along with the CYP activity of the supernatant ( C ) and the 14 C heme counts in MYC Ab pulldowns of CYP3A4 from the supernatants ( D ). Data are the mean ± SD of three experiments. ∗∗∗ p < 0.001, ∗∗ p < 0.01, one-way ANOVA. Ab, antibody; 14 C-Gly, 14 C-glycine; CYP, cytochrome P450; GlyA-CHO, glycine auxotroph Chinese hamster ovary cell; NO, nitric oxide; UT, untransfected cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Generated, Activity Assay, Expressing, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: Kinetics of NO-induced alterations in CYP450 activities and heme contents. GlyA-CHO cells that were transfected to express CYP2D6 or 3A4 were incubated with 14 C-Gly to generate 14 C-labeled heme. Protein synthesis was then blocked with Chx, and NOC18 was added at concentrations of 0 ( red ), 5 (green), and 100 ( blue ) μM, followed by harvesting and analysis of the cell supernatants at designated time points. A and C, 14 C-heme counts in CYP2D6 and CYP3A4 MYC Ab pull-downs. B and D, CYP2D6 and CYP3A4 activities. Data are the mean ± SD; n = 3 experiments. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ns = not significant, one-way ANOVA. Ab, antibody; 14 C-Gly, 14 C-glycine; Chx, cycloheximide; CYP450, cytochrome P450; GlyA-CHO, glycine auxotroph Chinese hamster ovary cell; NO, nitric oxide; NOC18, 2,2′-(hydroxynitrosohydrazino)bis-ethanolamine; UT, untransfected cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Transfection, Incubation, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: NO stimulates cell heme insertion into CYP3A4 by a GAPDH-dependent mechanism. GlyA-CHO cells that had been subjected to siRNA knockdown of GAPDH expression or treated with scrambled siRNA were administered 14 C-Gly and subsequently transfected to express CYP3A4 either alone or in conjunction with siRNA-resistant versions of wildtype HA-GAPDH or the heme-binding defective HA-GAPDH-H53A variant. Cells were subsequently treated with 0 or 5 μM NOC18, and after 6 h, the cell supernatants were prepared, and their CYP3A4 14 C-heme levels ( A ) and the activities ( B ) were assessed. Data are the mean ± SD; n = 3 experiments. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ns, not significant, one-way ANOVA. 14 C-Gly, 14 C-glycine; CYP, cytochrome P450; GlyA-CHO, glycine auxotroph Chinese hamster ovary cell; HA, hemagglutinin; NO, nitric oxide; NOC18, 2,2′-(hydroxynitrosohydrazino)bis-ethanolamine; UT, untransfected cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Knockdown, Expressing, Transfection, Binding Assay, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: Effect of NOC18 exposures on Hsp90 association with CYP3A4. GlyA-CHO cells that had been transfected to express FLAG- and MYC-tagged CYP3A4 in media enriched with 14 C-Gly were given Chx and given NOC18 at the designated concentrations, cultured for 6 h, harvested, and the cell supernatants underwent MYC Ab pulldown and Western analysis to compare bound Hsp90 levels. A, representative Western blot indicating the relative levels of Hsp90 associated with CYP3A4. B, corresponding normalized band intensities. Data are the mean ± SD of three independent experiments, ∗∗ p < 0.01, one-way ANOVA. Ab, antibody; 14 C-Gly, 14 C-glycine; Chx, cycloheximide; CYP, cytochrome P450; GlyA-CHO, glycine auxotroph Chinese hamster ovary cell; Hsp90, heat shock protein 90; NOC18, 2,2′-(hydroxynitrosohydrazino)bis-ethanolamine; UT, untransfected cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Transfection, Cell Culture, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

doi: 10.1016/j.jbc.2025.110772

Figure Lengend Snippet: NO-driven heme insertion in CYP3A4 requires cell Hsp90 activity. GlyA-CHO cells underwent transfection to express CYP3A4 in the presence of 14 C-Gly. Cells then received 10 μM radicicol (an Hsp90 inhibitor) 1 h prior to the addition of 0 or 5 μM NOC18. The cells were harvested at the indicated times, and supernatants were analyzed to determine CYP3A4 14 C-heme content ( A ) and activity ( B ) at 6 h. Data are the mean ± SD; n = 3 experiments. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ns, not significant, one-way ANOVA. 14 C-Gly, 14 C-glycine; CYP, cytochrome P450; GlyA-CHO, glycine auxotroph Chinese hamster ovary cell; Hsp90, heat shock protein 90; NO, nitric oxide; NOC18, 2,2′-(hydroxynitrosohydrazino)bis-ethanolamine; UT, untransfected cell samples.

Article Snippet: The cell cultures were given 400 μM of SA (D1415; Sigma) for 2 to 3 days to deplete their endogenous heme pool, and when they reached approximately 70% confluency, they were transfected with 10 μg of FLAG- and MYC-tagged CYP3A4 (RC210170; Origene) or CYP2D6 (RC223749; Origene) expression plasmids using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Activity Assay, Transfection

Journal: Biochemistry and Biophysics Reports

Article Title: Cyp3a11 is not essential for the formation of murine bile acids

doi: 10.1016/j.bbrep.2017.02.011

Figure Lengend Snippet: Characterization of Cyp3a KO mice. (A-C) Body weight (A) and relative liver (B) and gallbladder (C) weight in female and male Cyp3a KO and wild-type mice. (D) Gene expression of Cyp3a11 in livers from female and male Cyp3a KO and wild-type mice. Mean values±SEM are plotted; n=5–11 mice or samples/group; * P<0.05, ** P<0.01, *** P<0.001**** P<0.0001 indicate differences between female Cyp3a KO and wild-type or male Cyp3a KO and wild-type mice analyzed with unpaired t -test.

Article Snippet: Mice with a deletion of 7 genes ( Cyp3a11 , Cyp3a16 , Cyp3a25 , Cyp3a41 , Cyp3a44 , Cyp3a57 , and Cyp3a59 ) in the Cyp3a gene cluster ( Cyp3a KO) were purchased from Taconic, Model 8841.

Techniques: Gene Expression

Journal: Biochemistry and Biophysics Reports

Article Title: Cyp3a11 is not essential for the formation of murine bile acids

doi: 10.1016/j.bbrep.2017.02.011

Figure Lengend Snippet: Bile acid composition in liver and gallbladder. Liver (pmol/mg tissue) and gallbladder (nmol/mg tissue) bile acids in female and male Cyp3a KO and wild type mice.

Article Snippet: Mice with a deletion of 7 genes ( Cyp3a11 , Cyp3a16 , Cyp3a25 , Cyp3a41 , Cyp3a44 , Cyp3a57 , and Cyp3a59 ) in the Cyp3a gene cluster ( Cyp3a KO) were purchased from Taconic, Model 8841.

Techniques:

Journal: Biochemistry and Biophysics Reports

Article Title: Cyp3a11 is not essential for the formation of murine bile acids

doi: 10.1016/j.bbrep.2017.02.011

Figure Lengend Snippet: Expression of genes involved in bile acid synthesis. (A-D) Expression of cytochrome P450 genes Cyp7a1 (A), Cyp8b1 (B), Cyp27a1 (C) and Cyp7b1 (D) in livers from female and male Cyp3a KO and wild-type mice. Mean values±SEM are plotted; n=5–11 mice or samples/group; * P<0.05, ** P<0.01, *** P<0.001**** P<0.0001 indicate differences between female Cyp3a KO and wild-type or male Cyp3a KO and wild-type mice analyzed with unpaired t -test.

Article Snippet: Mice with a deletion of 7 genes ( Cyp3a11 , Cyp3a16 , Cyp3a25 , Cyp3a41 , Cyp3a44 , Cyp3a57 , and Cyp3a59 ) in the Cyp3a gene cluster ( Cyp3a KO) were purchased from Taconic, Model 8841.

Techniques: Expressing

Journal: Molecular pharmaceutics

Article Title: Transfected MDCK cell line with enhanced expression of CYP3A4 and P-glycoprotein as a model to study their role in drug transport and metabolism.

doi: 10.1021/mp200487h

Figure Lengend Snippet: Figure 1. Expression of CYP3A4: (1) molecular weight markers; (2) MDCK-MDR1 cells; (3) Caco-2 cells; (4) MDCK-MDR1 cells transfected with CYP3A4.

Article Snippet: We obta ined human CYP3A4 cDNA (NM_017460.3) insert as 10 μg of transfection-ready plasmid (pCMV6-Neo- CYP3A4) from Origene Technologies.

Techniques: Expressing, Molecular Weight, Transfection

Journal: Molecular pharmaceutics

Article Title: Transfected MDCK cell line with enhanced expression of CYP3A4 and P-glycoprotein as a model to study their role in drug transport and metabolism.

doi: 10.1021/mp200487h

Figure Lengend Snippet: Figure 2. Immunoblot showing 56.5 kDa band for CYP3A4 protein: (lane 1) molecular marker; (lane 2) MDCK-WT; (lane 3) MDCK- WT-CYP3A4; (lane 4) MDCK-MDR1; (lane 5) MDCK-MDR1- CYP3A4; (lane 6) blank; (lanes 7−9) human intestinal microsomes. Lanes 2−5 were loaded with 20 μg of microsomal protein whereas lanes 7−9 were loaded with 10, 5, and 2.5 μg of microsomal protein respectively.

Article Snippet: We obta ined human CYP3A4 cDNA (NM_017460.3) insert as 10 μg of transfection-ready plasmid (pCMV6-Neo- CYP3A4) from Origene Technologies.

Techniques: Western Blot, Marker

Journal: Molecular pharmaceutics

Article Title: Transfected MDCK cell line with enhanced expression of CYP3A4 and P-glycoprotein as a model to study their role in drug transport and metabolism.

doi: 10.1021/mp200487h

Figure Lengend Snippet: Figure 4. Comparative CYP3A4 mRNA levels in MDCK-WT, MDCK-MDR1, MDCK-WT-CYP3A4, MDCK-MDR1-CYP3A4, Caco-2 and HepG2 cells after CYP3A4-NEO transfection. All data were presented as mean ± SD. * indicates statistical significance at p < 0.05 with n = 6.

Article Snippet: We obta ined human CYP3A4 cDNA (NM_017460.3) insert as 10 μg of transfection-ready plasmid (pCMV6-Neo- CYP3A4) from Origene Technologies.

Techniques: Transfection

Journal: Molecular pharmaceutics

Article Title: Transfected MDCK cell line with enhanced expression of CYP3A4 and P-glycoprotein as a model to study their role in drug transport and metabolism.

doi: 10.1021/mp200487h

Figure Lengend Snippet: Figure 5. Flourescent metabolism assay for determining CYP3A4 functional activity in the transfected cells. All data were presented as mean ± SD. * indicates statistical significance at p < 0.05 with n = 6.

Article Snippet: We obta ined human CYP3A4 cDNA (NM_017460.3) insert as 10 μg of transfection-ready plasmid (pCMV6-Neo- CYP3A4) from Origene Technologies.

Techniques: Functional Assay, Activity Assay, Transfection

Journal: Molecular pharmaceutics

Article Title: Transfected MDCK cell line with enhanced expression of CYP3A4 and P-glycoprotein as a model to study their role in drug transport and metabolism.

doi: 10.1021/mp200487h

Figure Lengend Snippet: Figure 6. Determination of IC50 value for inhibition of CYP3A4- mediated metabolism of Vivid by ketoconazole in MDCK-MDR1- CYP3A4 cells in 96-well plates. Various concentrations of ketoconazole were incubated in the presence of 20 μM Vivid for 40 min. Values are expressed as the % of control rates (absence of inhibitor).

Article Snippet: We obta ined human CYP3A4 cDNA (NM_017460.3) insert as 10 μg of transfection-ready plasmid (pCMV6-Neo- CYP3A4) from Origene Technologies.

Techniques: Inhibition, Incubation, Control

Journal: Molecular pharmaceutics

Article Title: Transfected MDCK cell line with enhanced expression of CYP3A4 and P-glycoprotein as a model to study their role in drug transport and metabolism.

doi: 10.1021/mp200487h

Figure Lengend Snippet: Figure 7. (a) Transport of 200 μM cortisol in MDCK-WT and MDCK-MDR1 cells alone and transfected with CYP3A4. (b) Time dependent formation of CYP3A4-mediated metabolite of cortisol (6β-hydroxy cortisol). n = 6 ± SD.

Article Snippet: We obta ined human CYP3A4 cDNA (NM_017460.3) insert as 10 μg of transfection-ready plasmid (pCMV6-Neo- CYP3A4) from Origene Technologies.

Techniques: Transfection

Journal: Molecular pharmaceutics

Article Title: Transfected MDCK cell line with enhanced expression of CYP3A4 and P-glycoprotein as a model to study their role in drug transport and metabolism.

doi: 10.1021/mp200487h

Figure Lengend Snippet: Figure 8. Transport of 200 μM cortisol alone or in the presence of morphine (3 μM) or naringin (50 μM) across (A) MDCK-WT, (B) MDCK- MDR1, (C) MDCK-CYP3A4, (D) MDCK-MDR1-CYP3A4 cell lines. n = 4 ± SD.

Article Snippet: We obta ined human CYP3A4 cDNA (NM_017460.3) insert as 10 μg of transfection-ready plasmid (pCMV6-Neo- CYP3A4) from Origene Technologies.

Techniques:

Journal: Molecular pharmaceutics

Article Title: Transfected MDCK cell line with enhanced expression of CYP3A4 and P-glycoprotein as a model to study their role in drug transport and metabolism.

doi: 10.1021/mp200487h

Figure Lengend Snippet: Figure 9. Permeabillity values for transport of 200 μM cortisol alone or in the presence of morphine (3 μM) or naringin (50 μM) across MDCK-WT and MDCK-MDR1 cells alone and transfected with CYP3A4. * indicates statistical significance at p < 0.05 with n = 4 when compared to transport in wt cell line for that combination. ¥ indicates statistical significance at p < 0.05 with n = 4 when compared to transport of cortisol in that respective cell line.

Article Snippet: We obta ined human CYP3A4 cDNA (NM_017460.3) insert as 10 μg of transfection-ready plasmid (pCMV6-Neo- CYP3A4) from Origene Technologies.

Techniques: Transfection

Journal: British Journal of Pharmacology

Article Title: UDP‐Glucuronosyltransferase (UGT)‐mediated attenuations of cytochrome P450 3A4 activity: UGT isoform‐dependent mechanism of suppression

doi: 10.1111/bph.14900

Figure Lengend Snippet: Immunoblotting to confirm the expression of the recombinant enzymes, CYP3A4, CPR, UGT1A9, and UGT2B7. Antibodies against human‐CYP3A, human‐CPR, human‐UGT1A, human‐UGT2B7, and human‐GAPDH were used as primary antibodies. HLM and recombinant enzymes were positive controls. The protein amounts loaded were as follows: HLM, 10 μg; Supersomes (as a CYP3A4 standard), 5 pmol of CYP3A4; purified CPR, 100 ng; and the Sf9 homogenates (as denoted), 20 μg, respectively. Mock represents homogenates prepared from Sf9 cells infected with control virus

Article Snippet: The blotted membrane was washed with TBS‐T, 20‐mM Tris‐HCl (pH 7.5) containing 150‐mM NaCl, and 0.1% Tween 20 and blocked with 2% skim milk in TBS‐T at room temperature for 30 min. For detection of targets, the following primary antibodies and detection kit were utilized: rabbit polyclonal anti‐UGT1A antibody (H‐300, Cat# sc‐25847, RRID:AB_2241481), mouse monoclonal anti‐CPR antibody (F‐10, Cat# sc‐25270, RRID:AB_627391; Santa Cruz Biotechnology, Dallas, TX), goat polyclonal anti‐CYP3A2 serum (Daiichi Pure Chemicals, Tokyo, Japan; Cat# 877), rabbit polyclonal anti‐UGT2B7 antibody, rabbit polyclonal anti‐GAPDH antibody (Proteintech, Rosemont, IL; respective Cat# 16661‐1‐AP and 10494‐1‐AP, respective RRID:AB_2214249 and AB_2263076), rabbit polyclonal anti‐calnexin (CNX) antibody (GeneTex, Irvine, CA; Cat# GTX112886, RRID:AB_1949822), rabbit polyclonal anti‐6‐His antibody (Bethyl Laboratories, Montgomery, TX; Cat# A190‐114A, RRID:AB_67321), rabbit polyclonal anti‐hemagglutinin (HA) antibody (Sigma‐Aldrich, St. Louis, MO; Cat# H6908, RRID:AB_260070), and WB‐MAB‐3A Human CYP3A Western Blotting Kit (Corning Gentest, Woburm, MA; Cat# 458254, RRID:AB_712943).

Techniques: Western Blot, Expressing, Recombinant, Purification, Infection, Control, Virus

Journal: British Journal of Pharmacology

Article Title: UDP‐Glucuronosyltransferase (UGT)‐mediated attenuations of cytochrome P450 3A4 activity: UGT isoform‐dependent mechanism of suppression

doi: 10.1111/bph.14900

Figure Lengend Snippet: Comparison of CYP3A4 activity in the absence and presence of UGT1A9/2B7. Sf9 cells were seeded in 35‐mm dishes and independently infected with recombinant baculoviruses. CYP3A4 activity was measured with Sf9 homogenates (20‐μg protein) as enzyme source and luciferin‐PFBE (50 μM) as a substrate. The mean ± SD of CYP3A4 activities are shown (N = 6). The result of quantitative immunoblotting is shown in Figure S1, and protein levels of expressed enzymes are summarized in Table S1 (a). Kinetic analysis to estimate the effect of coexpression of UGT1A9 and UGT2B7 on CYP3A4 activity. Using Sf9 homogenates as enzyme source, CYP3A4 activity was compared between CYP3A4/CPR homogenates (none, open circle) and CYP3A4/CPR/UGT homogenates (UGT1A9, grey triangle; UGT2B7, closed square). Data were fitted to the Michaelis–Menten model. Each plot represents the mean ± SD of six different samples (b). Calculated Vmax and K M values were compared. The kinetic parameters were determined in each sample, to obtain six sets of Vmax and K M values in each group. The mean ± SD of six sets of parameters are shown, and the values are listed in Table ​Table11 (c). Statistical significance was determined by ANOVA followed by Dunnett's test only when the former F value was significant and there was no significant variance in homogeneity (vs. none)

Article Snippet: The blotted membrane was washed with TBS‐T, 20‐mM Tris‐HCl (pH 7.5) containing 150‐mM NaCl, and 0.1% Tween 20 and blocked with 2% skim milk in TBS‐T at room temperature for 30 min. For detection of targets, the following primary antibodies and detection kit were utilized: rabbit polyclonal anti‐UGT1A antibody (H‐300, Cat# sc‐25847, RRID:AB_2241481), mouse monoclonal anti‐CPR antibody (F‐10, Cat# sc‐25270, RRID:AB_627391; Santa Cruz Biotechnology, Dallas, TX), goat polyclonal anti‐CYP3A2 serum (Daiichi Pure Chemicals, Tokyo, Japan; Cat# 877), rabbit polyclonal anti‐UGT2B7 antibody, rabbit polyclonal anti‐GAPDH antibody (Proteintech, Rosemont, IL; respective Cat# 16661‐1‐AP and 10494‐1‐AP, respective RRID:AB_2214249 and AB_2263076), rabbit polyclonal anti‐calnexin (CNX) antibody (GeneTex, Irvine, CA; Cat# GTX112886, RRID:AB_1949822), rabbit polyclonal anti‐6‐His antibody (Bethyl Laboratories, Montgomery, TX; Cat# A190‐114A, RRID:AB_67321), rabbit polyclonal anti‐hemagglutinin (HA) antibody (Sigma‐Aldrich, St. Louis, MO; Cat# H6908, RRID:AB_260070), and WB‐MAB‐3A Human CYP3A Western Blotting Kit (Corning Gentest, Woburm, MA; Cat# 458254, RRID:AB_712943).

Techniques: Comparison, Activity Assay, Infection, Recombinant, Western Blot

Journal: British Journal of Pharmacology

Article Title: UDP‐Glucuronosyltransferase (UGT)‐mediated attenuations of cytochrome P450 3A4 activity: UGT isoform‐dependent mechanism of suppression

doi: 10.1111/bph.14900

Figure Lengend Snippet: Pull‐down assay to detect protein–protein interactions between CYP3A4 and UGT1A9 WT/ΔTM. CYP3A4‐6 × His and UGT1A9‐HA were transiently expressed in COS‐1 cells, and lysates were prepared. Pull‐down assay was performed by mixing Ni‐nitrilotriacetic acid magnetic beads and 600 μg of lysate. Bound proteins were eluted by buffer containing a high level of imidazole. Pull‐down samples and 20‐μg lysates (3% input) were analysed by immunoblotting

Article Snippet: The blotted membrane was washed with TBS‐T, 20‐mM Tris‐HCl (pH 7.5) containing 150‐mM NaCl, and 0.1% Tween 20 and blocked with 2% skim milk in TBS‐T at room temperature for 30 min. For detection of targets, the following primary antibodies and detection kit were utilized: rabbit polyclonal anti‐UGT1A antibody (H‐300, Cat# sc‐25847, RRID:AB_2241481), mouse monoclonal anti‐CPR antibody (F‐10, Cat# sc‐25270, RRID:AB_627391; Santa Cruz Biotechnology, Dallas, TX), goat polyclonal anti‐CYP3A2 serum (Daiichi Pure Chemicals, Tokyo, Japan; Cat# 877), rabbit polyclonal anti‐UGT2B7 antibody, rabbit polyclonal anti‐GAPDH antibody (Proteintech, Rosemont, IL; respective Cat# 16661‐1‐AP and 10494‐1‐AP, respective RRID:AB_2214249 and AB_2263076), rabbit polyclonal anti‐calnexin (CNX) antibody (GeneTex, Irvine, CA; Cat# GTX112886, RRID:AB_1949822), rabbit polyclonal anti‐6‐His antibody (Bethyl Laboratories, Montgomery, TX; Cat# A190‐114A, RRID:AB_67321), rabbit polyclonal anti‐hemagglutinin (HA) antibody (Sigma‐Aldrich, St. Louis, MO; Cat# H6908, RRID:AB_260070), and WB‐MAB‐3A Human CYP3A Western Blotting Kit (Corning Gentest, Woburm, MA; Cat# 458254, RRID:AB_712943).

Techniques: Pull Down Assay, Protein-Protein interactions, Magnetic Beads, Western Blot

Journal: British Journal of Pharmacology

Article Title: UDP‐Glucuronosyltransferase (UGT)‐mediated attenuations of cytochrome P450 3A4 activity: UGT isoform‐dependent mechanism of suppression

doi: 10.1111/bph.14900

Figure Lengend Snippet: Effect of C‐terminal truncated mutants of UGT1A9 and UGT2B7 on CYP3A4 activity. CYP3A4 activity was measured with Sf9 homogenates (20‐μg protein) as enzyme source and luciferin‐PFBE (50 μM) as a substrate. The mean ± SD of CYP3A4 activities are shown (N = 6). The result of quantitative immunoblotting is shown in Figure S2, and protein levels of expressed enzymes are summarized in Table S2 (a). Kinetic analysis showing the effect of UGT truncated mutants. The homogenates mentioned above were used as enzyme sources, and CYP3A4 activity was compared between CYP3A4/CPR homogenates (none, open circle) and CYP3A4/CPR/UGT homogenates (UGT1A9 ΔTM, grey triangle; UGT2B7 ΔTM, closed square). Data were fitted to the Michaelis–Menten model. Each plot represents the mean ± SD of the six samples (b). Calculated Vmax and K M values were compared. The kinetic parameters were determined in each sample, so we obtained six pairs of Vmax and K M values in each group. The mean ± SD of six calculated parameters are shown, and the values are listed in Table ​Table33 (c). Statistical significance was determined by ANOVA followed by Dunnett's test only when the former F value was significant and there was no significant variance in homogeneity (vs. none)

Article Snippet: The blotted membrane was washed with TBS‐T, 20‐mM Tris‐HCl (pH 7.5) containing 150‐mM NaCl, and 0.1% Tween 20 and blocked with 2% skim milk in TBS‐T at room temperature for 30 min. For detection of targets, the following primary antibodies and detection kit were utilized: rabbit polyclonal anti‐UGT1A antibody (H‐300, Cat# sc‐25847, RRID:AB_2241481), mouse monoclonal anti‐CPR antibody (F‐10, Cat# sc‐25270, RRID:AB_627391; Santa Cruz Biotechnology, Dallas, TX), goat polyclonal anti‐CYP3A2 serum (Daiichi Pure Chemicals, Tokyo, Japan; Cat# 877), rabbit polyclonal anti‐UGT2B7 antibody, rabbit polyclonal anti‐GAPDH antibody (Proteintech, Rosemont, IL; respective Cat# 16661‐1‐AP and 10494‐1‐AP, respective RRID:AB_2214249 and AB_2263076), rabbit polyclonal anti‐calnexin (CNX) antibody (GeneTex, Irvine, CA; Cat# GTX112886, RRID:AB_1949822), rabbit polyclonal anti‐6‐His antibody (Bethyl Laboratories, Montgomery, TX; Cat# A190‐114A, RRID:AB_67321), rabbit polyclonal anti‐hemagglutinin (HA) antibody (Sigma‐Aldrich, St. Louis, MO; Cat# H6908, RRID:AB_260070), and WB‐MAB‐3A Human CYP3A Western Blotting Kit (Corning Gentest, Woburm, MA; Cat# 458254, RRID:AB_712943).

Techniques: Activity Assay, Western Blot

Journal: British Journal of Pharmacology

Article Title: UDP‐Glucuronosyltransferase (UGT)‐mediated attenuations of cytochrome P450 3A4 activity: UGT isoform‐dependent mechanism of suppression

doi: 10.1111/bph.14900

Figure Lengend Snippet: Effect of dexamethasone treatment on CYP3A–UGT1A interaction in rat liver. Induction ranges of CYP3As, UGT1As, and CPR were determined by immunoblotting. Wistar rats (male, 5 weeks old, N = 5 in each group) were treated with dexamethasone in saline at a dose of 80 mg·kg−1 in a day for 4 days. The control group was treated with the same amount of saline. One day after the last treatment, liver microsomes were prepared and analysed individually. Dex represents dexamethasone‐treated rats. Calnexin (CNX), an ER marker protein, was also detected as a loading control. Relative protein levels (control mean = 1.0) are shown as the mean ± SD. The amount of microsomes utilized in the immunoblotting was 10 μg except for detection of CYP3As in dexamethasone‐treated rats (1 μg) (a). CYP3A turnovers are shown as the mean ± SD (N = 5) for each substrate concentration. Kinetic analysis with individual rat liver microsomes. Data were fitted to the Michaelis–Menten model. Values of the y axis represent turnover of CYP3A, an activity related to CYP3A levels as determined by immunoblotting shown in Figure ​Figure5a,5a, not an activity per microsomal protein (b). Five sets of Vmax and K M for each group were calculated. These are shown as the mean ± SD. Statistical significance was determined by Student's t test (unpaired, two tailed) (c)

Article Snippet: The blotted membrane was washed with TBS‐T, 20‐mM Tris‐HCl (pH 7.5) containing 150‐mM NaCl, and 0.1% Tween 20 and blocked with 2% skim milk in TBS‐T at room temperature for 30 min. For detection of targets, the following primary antibodies and detection kit were utilized: rabbit polyclonal anti‐UGT1A antibody (H‐300, Cat# sc‐25847, RRID:AB_2241481), mouse monoclonal anti‐CPR antibody (F‐10, Cat# sc‐25270, RRID:AB_627391; Santa Cruz Biotechnology, Dallas, TX), goat polyclonal anti‐CYP3A2 serum (Daiichi Pure Chemicals, Tokyo, Japan; Cat# 877), rabbit polyclonal anti‐UGT2B7 antibody, rabbit polyclonal anti‐GAPDH antibody (Proteintech, Rosemont, IL; respective Cat# 16661‐1‐AP and 10494‐1‐AP, respective RRID:AB_2214249 and AB_2263076), rabbit polyclonal anti‐calnexin (CNX) antibody (GeneTex, Irvine, CA; Cat# GTX112886, RRID:AB_1949822), rabbit polyclonal anti‐6‐His antibody (Bethyl Laboratories, Montgomery, TX; Cat# A190‐114A, RRID:AB_67321), rabbit polyclonal anti‐hemagglutinin (HA) antibody (Sigma‐Aldrich, St. Louis, MO; Cat# H6908, RRID:AB_260070), and WB‐MAB‐3A Human CYP3A Western Blotting Kit (Corning Gentest, Woburm, MA; Cat# 458254, RRID:AB_712943).

Techniques: Western Blot, Saline, Control, Marker, Concentration Assay, Activity Assay, Two Tailed Test

Journal: British Journal of Pharmacology

Article Title: UDP‐Glucuronosyltransferase (UGT)‐mediated attenuations of cytochrome P450 3A4 activity: UGT isoform‐dependent mechanism of suppression

doi: 10.1111/bph.14900

Figure Lengend Snippet: Effect of dexamethasone treatment on hepatic CYP3A activity in Wistar rat

Article Snippet: The blotted membrane was washed with TBS‐T, 20‐mM Tris‐HCl (pH 7.5) containing 150‐mM NaCl, and 0.1% Tween 20 and blocked with 2% skim milk in TBS‐T at room temperature for 30 min. For detection of targets, the following primary antibodies and detection kit were utilized: rabbit polyclonal anti‐UGT1A antibody (H‐300, Cat# sc‐25847, RRID:AB_2241481), mouse monoclonal anti‐CPR antibody (F‐10, Cat# sc‐25270, RRID:AB_627391; Santa Cruz Biotechnology, Dallas, TX), goat polyclonal anti‐CYP3A2 serum (Daiichi Pure Chemicals, Tokyo, Japan; Cat# 877), rabbit polyclonal anti‐UGT2B7 antibody, rabbit polyclonal anti‐GAPDH antibody (Proteintech, Rosemont, IL; respective Cat# 16661‐1‐AP and 10494‐1‐AP, respective RRID:AB_2214249 and AB_2263076), rabbit polyclonal anti‐calnexin (CNX) antibody (GeneTex, Irvine, CA; Cat# GTX112886, RRID:AB_1949822), rabbit polyclonal anti‐6‐His antibody (Bethyl Laboratories, Montgomery, TX; Cat# A190‐114A, RRID:AB_67321), rabbit polyclonal anti‐hemagglutinin (HA) antibody (Sigma‐Aldrich, St. Louis, MO; Cat# H6908, RRID:AB_260070), and WB‐MAB‐3A Human CYP3A Western Blotting Kit (Corning Gentest, Woburm, MA; Cat# 458254, RRID:AB_712943).

Techniques: Activity Assay, Control

Journal: British Journal of Pharmacology

Article Title: UDP‐Glucuronosyltransferase (UGT)‐mediated attenuations of cytochrome P450 3A4 activity: UGT isoform‐dependent mechanism of suppression

doi: 10.1111/bph.14900

Figure Lengend Snippet: Postulated mechanism of UGT‐mediated suppression of CYP3A4 activity

Article Snippet: The blotted membrane was washed with TBS‐T, 20‐mM Tris‐HCl (pH 7.5) containing 150‐mM NaCl, and 0.1% Tween 20 and blocked with 2% skim milk in TBS‐T at room temperature for 30 min. For detection of targets, the following primary antibodies and detection kit were utilized: rabbit polyclonal anti‐UGT1A antibody (H‐300, Cat# sc‐25847, RRID:AB_2241481), mouse monoclonal anti‐CPR antibody (F‐10, Cat# sc‐25270, RRID:AB_627391; Santa Cruz Biotechnology, Dallas, TX), goat polyclonal anti‐CYP3A2 serum (Daiichi Pure Chemicals, Tokyo, Japan; Cat# 877), rabbit polyclonal anti‐UGT2B7 antibody, rabbit polyclonal anti‐GAPDH antibody (Proteintech, Rosemont, IL; respective Cat# 16661‐1‐AP and 10494‐1‐AP, respective RRID:AB_2214249 and AB_2263076), rabbit polyclonal anti‐calnexin (CNX) antibody (GeneTex, Irvine, CA; Cat# GTX112886, RRID:AB_1949822), rabbit polyclonal anti‐6‐His antibody (Bethyl Laboratories, Montgomery, TX; Cat# A190‐114A, RRID:AB_67321), rabbit polyclonal anti‐hemagglutinin (HA) antibody (Sigma‐Aldrich, St. Louis, MO; Cat# H6908, RRID:AB_260070), and WB‐MAB‐3A Human CYP3A Western Blotting Kit (Corning Gentest, Woburm, MA; Cat# 458254, RRID:AB_712943).

Techniques: Activity Assay