Journal: Foods

Article Title: Ethanol Extract of Mao Jian Green Tea Attenuates Gastrointestinal Symptoms in a Rat Model of Irritable Bowel Syndrome with Constipation via the 5-hydroxytryptamine Signaling Pathway

doi: 10.3390/foods12051101

Figure Lengend Snippet: Western blotting analysis of TPH1 ( A ), TPH2 ( B ), SERT ( C ), 5-HT 3 receptors ( D ), 5-HT 4 receptors ( E ), CaM ( F ), and MLCK ( G ) in colonic tissues of each group. Capital and lowercase letters above the bar indicate the difference significance at the 0.01 or 0.05 levels, respectively. ( n = 8 for each group). NC: negative control group; MG: model group, MSP: mosapride group, MJGT_EE: Mao Jian Green Tea ethanol extract group; 5-HT: 5-hydroxytryptamine; TPH1: tryptophan hydroxylase 1; TPH2: tryptophan hydroxylase 2; SERT: serotonin transporter; CAM: calmodulin; MLCK: myosin light chain kinase; 5-HT 3 R: 5-HT 3 receptor; 5-HT 4 R: 5-HT 4 receptor.

Article Snippet: Second, primary antibodies against 5-hydroxytryptamine receptor 3 (5-HT 3 R; bs-2126R), 5-hydroxytryptamine 4 receptor (5-HT 4 R; bs-12054R), the serotonin transporter (SERT, bs-1893R), tryptophan hydroxylase 1 (TPH1; bs-1215R), tryptophan hydroxylase 2 (TPH2; bs-8729R), calmodulin (CaM; bs-3666R), or myosin light chain kinase (MLCK) (Bioss Biotechnology Co., Ltd., Beijing, China) were added (1:1000) to the membrane and incubated at 4 °C for 12 h. Next, the membrane was washed thrice with TBST buffer (T1081, Solarbio Science & Technology Co., Ltd., Beijing, China); then, secondary antibodies (HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) or HRP-conjugated Affinipure Goat Anti-Rabbit IgGs (H+L); 1:5000) were added to the membrane and incubated at room temperature for 1 h. Finally, at the end of the incubation, the membrane was again washed thrice with the TBST buffer, developed, exposed in a dark room, and imaged using a ChemiDoc MP Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Techniques: Western Blot, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Prenatal Opioid and Alcohol Exposures: Association with Altered Placental Serotonin Transporter Structure and/or Expression

doi: 10.3390/ijms252111570

Figure Lengend Snippet: Effect of in utero opioid exposure on SERT and ABCB1 expression in human placental membranous vesicles. ( A ) Downregulation of SERT levels in qWestern blot analysis of protein lysates, comparing 20 first and second trimester opioid-exposed human placentas with 20 unexposed controls individually matched for fetal sex, GA, and maternal age. ( B ) Reduced levels of the drug transporter ABCB1 in opioid-exposed placentas. Inside out placental brush border membrane vesicles were used to measure and quantify the ABCB1 expression. In both ( A , B ), Grb2 served as a loading control. Data are presented as fold change (* for p < 0.05, *** for p < 0.001).

Article Snippet: SLC6A4 antibody to C-terminus SERT (OAEB02575) was from Aviva Systems Biology, Corp. (San Diego, CA, USA).

Techniques: In Utero, Expressing, Membrane, Control

Journal: International Journal of Molecular Sciences

Article Title: Prenatal Opioid and Alcohol Exposures: Association with Altered Placental Serotonin Transporter Structure and/or Expression

doi: 10.3390/ijms252111570

Figure Lengend Snippet: SERT is modified by maternal opioid exposure. ( A ) Immunoblot detection of SERT in placental vesicles of first and second trimester fetuses, using a rabbit polyclonal anti-serotonin transporter antibody specific for 70 kDa SERT. Grb2 served as a loading control. Subjects who admitted to chronic opioid use were compared to fetal sex-, GA-, and maternal age-matched controls. Left panel , lanes 1–4 are unexposed controls. Lanes 5–8 are subjects not exposed to opioids or EtOH but who were being treated for depression with SSRIs. Right panel , 12 opiod-exposed cases not exposed to other drugs. ( B ) Alteration of the SERT protein band pattern is seen only in opioid-exposed cases (lanes 4–10), not with other drug exposures—Adderall (lanes 1–3) and Keppra (lanes 11–12). The cleaved forms of SERT were observed only in opiate cases (lanes 4–10, top panel). ( C ) The smaller of the double-bands between 34 and 32 kDA representing cleaved forms of SERT (clSERT) in the opioid-exposed cases may represent a hypo-phosphorylated SERT fragment (see ).

Article Snippet: SLC6A4 antibody to C-terminus SERT (OAEB02575) was from Aviva Systems Biology, Corp. (San Diego, CA, USA).

Techniques: Modification, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Prenatal Opioid and Alcohol Exposures: Association with Altered Placental Serotonin Transporter Structure and/or Expression

doi: 10.3390/ijms252111570

Figure Lengend Snippet: Cleaved SERT isoforms in opioid-exposed cases are within the central domain . ( A ) The immunoblot used for was used for detection of the central domain and C-terminal of SERT in placental vesicles from first and second trimester human pregnancies. Western immunoblots demonstrate expression of SERT isoforms in vesicles from 12-opioid exposed placentas at two GAs. Post-translational modification of SERT was studied by treatment with phosphatase inhibitors (Lane 5 vs. 6). This resulted in enhancement of the lower of the opioid-induced bands at 32–34 kDa, suggesting that most of this smaller clSERT is unphosphorylated. Antibodies to the N-terminal of SERT did not recognize either of the smaller (32 and 34 kDa) bands, while an antibody to the C-terminal region of SERT recognized both bands (upper panel), identical to the bands identified by antibodies to the central region of SERT (lower panel). SDS-PAGE stained with Coomassie blue was used to assure equal protein loading. The actin band (42 kDa) is labeled. ( B ) An antibody specific for the macrophage marker CD163 was used as a negative control, in combination with an antibody to GAPDH as a positive control, to confirm the purity of placental vesicles, the lack of contamination by other sources of SERT, and the specificity of the SERT double bands in panels ( A , B ) Note the absence of a band at 70 kDa.

Article Snippet: SLC6A4 antibody to C-terminus SERT (OAEB02575) was from Aviva Systems Biology, Corp. (San Diego, CA, USA).

Techniques: Western Blot, Expressing, Modification, SDS Page, Staining, Labeling, Marker, Negative Control, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Prenatal Opioid and Alcohol Exposures: Association with Altered Placental Serotonin Transporter Structure and/or Expression

doi: 10.3390/ijms252111570

Figure Lengend Snippet: SERT sequencing in opioid cases. The Edman degradation experiment was performed to get the N-terminal sequence of truncated SERT protein double-band versions in opioid-exposed placental vesicles. Two SERT fragments were present in the opioid samples. The sequences were compared with two 5HT transporter isoforms downloaded from Unipro. Sequence alignment was performed using ClustalW2 on two isoforms of SERT fragments on SLC6A4 (Synonyms:HTT, SERT; Organism Homo sapiens (Human), Taxonomic identifier9606 [NCBI]). The first four amino acids of the Central region of SERT were detected in two potential truncated bands corresponding to the sequence of SERT. In combination with the western blot results, we confirm the presence of a truncated SERT protein. Truncated proteins were located using the sequence alignment tool. Version 1 (the upper band) encompasses the amino acids 183–227 (out of 672) of human SERT. Truncated Version 2 (lower band) encompasses amino acids 224–276 of human SERT, which includes the phosphorylation site Thr276.

Article Snippet: SLC6A4 antibody to C-terminus SERT (OAEB02575) was from Aviva Systems Biology, Corp. (San Diego, CA, USA).

Techniques: Sequencing, Western Blot, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Prenatal Opioid and Alcohol Exposures: Association with Altered Placental Serotonin Transporter Structure and/or Expression

doi: 10.3390/ijms252111570

Figure Lengend Snippet: Prenatal alcohol exposure-associated downregulation of SERT and ABCB1 in placenta vesicles, placenta-derived exosomes, and fetal brain-derived exosomes. ( A ) EtOH exposure is associated with downregulation of total SERT in right side out (brush border membranous) placental vesicles (IOV) and placenta-derived exosomes from maternal blood (PE; bars 3–4) from human placenta tissues (n = 20) obtained from first and second trimester pregnancies. Analysis was completed by ELISA using a SERT ELISA kit, and results were obtained in picograms/microliters according to SERT standards. Bars represent fold changes between SERT levels in vesicles from EtOH-exposed cases compared to unexposed controls. ( B ) A similar reduction in expression of ABCB1 was seen in inside-out vesicles IOV. ( C ) Downregulation of SERT in fetal brain-derived exosomes. All assays were completed in triplicate (catalog #HS0096, detection range is within 0, 0.5, 1.0, 2.5, 5.0, 10 ng/mL, or picogram/microliter, and sensitivity is 0.1 ng/mL). ( *** for p < 0.001).

Article Snippet: SLC6A4 antibody to C-terminus SERT (OAEB02575) was from Aviva Systems Biology, Corp. (San Diego, CA, USA).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Functional & Integrative Genomics

Article Title: Huang Qin decoction increases SLC6A4 expression and blocks the NFκB-mediated NLRP3/Caspase1/GSDMD pathway to disrupt colitis-associated carcinogenesis

doi: 10.1007/s10142-024-01334-x

Figure Lengend Snippet: HQD affects SLC6A4 expression and serotonin production in mouse colon tissues. (A) The ingredient-target-pathway/disease network of HQD in BATMAN-TCM. (B) The intersection of therapeutic targets screened in BATMAN-TCM and genes affected by Scutellaria baicalensis , Paeonia lactiflora , Glycyrrhiza uralensis , and Ziziphus jujuba in HERB 2.0. (C) Changes in SLC6A4 mRNA expression in AOM/DSS mice after HQD treatment detected by RT-qPCR. (D) Changes in SLC6A4 protein expression in AOM/DSS mice after HQD treatment detected by western blot assays. (E) Detection of serotonin (5-HT) production in mouse colon tissues by ELISA. The data are presented as the means ± SDs ( n = 6). *p < 0.05, ** p < 0.01, **** p < 0.0001. The data were analyzed by one-way ANOVA and Tukey’s multiple comparison test

Article Snippet: After being blocked with skim milk for 45 min at room temperature, the PVDF membranes were incubated overnight at 4 °C with primary antibodies against SLC6A4 (1:1000, H00006532-D01P, Novus Biological Inc., Littleton, CO, USA), p-NFκB p65 (1:1000, #3033, Cell Signaling Technologies, Beverly, MA, USA), NFκB p65 (1:1000, #8242, Cell Signaling Technologies), NLRP3 (1:1000, #15,101, Cell Signaling Technologies), Cleaved-Caspase-1 P20 (1:1000, PA5-99390, Invitrogen), Cleaved-GSDMD (1:1000, #50,928, Cell Signaling Technologies), GSDMD (1:1000, ab225867, Abcam, Cambridge, UK), Caspase1 (1:1000, ab138483, Abcam), ASC (1:1000, #67,824, Cell Signaling Technologies), and β-actin (1:200, ab115777, Abcam).

Techniques: Expressing, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Functional & Integrative Genomics

Article Title: Huang Qin decoction increases SLC6A4 expression and blocks the NFκB-mediated NLRP3/Caspase1/GSDMD pathway to disrupt colitis-associated carcinogenesis

doi: 10.1007/s10142-024-01334-x

Figure Lengend Snippet: The therapeutic effect of HQD on AOM/DSS mice is inhibited after SLC6A4 knockout. AOM/DSS was used to establish WT and SLC6A4-deficient mice, which were subsequently treated with HQD. (A) Changes in the body weight of mice. (B) DAI of the mice. (C) Length of colon tissue in mice. (D) Number of tumors in the colon tissues of the mice. (E) HE staining of pathological morphology of the colon tissues. (F) Detection of 5-HT, IL-1β and IL-18 levels in mouse colon tissues by ELISA. (G) The expression of the p-NFκB p65, NLRP3, Cleaved-Caspase1 P20, Cleaved-GSDMD, and ASC proteins was determined in the colon tissue of AOM/DSS mice by western blot assay. The data are presented as the means ± SDs ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The data were analyzed by an unpaired t-test or two-way ANOVA and Tukey’s multiple comparison test

Article Snippet: After being blocked with skim milk for 45 min at room temperature, the PVDF membranes were incubated overnight at 4 °C with primary antibodies against SLC6A4 (1:1000, H00006532-D01P, Novus Biological Inc., Littleton, CO, USA), p-NFκB p65 (1:1000, #3033, Cell Signaling Technologies, Beverly, MA, USA), NFκB p65 (1:1000, #8242, Cell Signaling Technologies), NLRP3 (1:1000, #15,101, Cell Signaling Technologies), Cleaved-Caspase-1 P20 (1:1000, PA5-99390, Invitrogen), Cleaved-GSDMD (1:1000, #50,928, Cell Signaling Technologies), GSDMD (1:1000, ab225867, Abcam, Cambridge, UK), Caspase1 (1:1000, ab138483, Abcam), ASC (1:1000, #67,824, Cell Signaling Technologies), and β-actin (1:200, ab115777, Abcam).

Techniques: Knock-Out, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Comparison

Journal: Functional & Integrative Genomics

Article Title: Huang Qin decoction increases SLC6A4 expression and blocks the NFκB-mediated NLRP3/Caspase1/GSDMD pathway to disrupt colitis-associated carcinogenesis

doi: 10.1007/s10142-024-01334-x

Figure Lengend Snippet: SLC6A4 downregulation promotes MODE-K cell pyroptosis treated with DS. MODE-K cells were infected with sh-SLC6A4 or sh-NC, pre-treated with DS, and induced with LPS-ATP. (A) Detection of SLC6A4 mRNA expression in MODE-K cells after infection by RT-qPCR. (B) MODE-K cell viability was examined using a CCK-8 assay. (C) Measurement of LDH release. (D) The expression of NLRP3, Cleaved-Caspase1 P20, and Cleaved-GSDMD in MODE-K cells was examined using a western blot. (E) Immunofluorescence detection of ASC expression in MODE-K cells. The data are presented as the means ± SD ( n = 3). ** p < 0.01, **** p < 0.0001. The data were analyzed by unpaired t-test or two-way ANOVA and Tukey’s multiple comparison test

Article Snippet: After being blocked with skim milk for 45 min at room temperature, the PVDF membranes were incubated overnight at 4 °C with primary antibodies against SLC6A4 (1:1000, H00006532-D01P, Novus Biological Inc., Littleton, CO, USA), p-NFκB p65 (1:1000, #3033, Cell Signaling Technologies, Beverly, MA, USA), NFκB p65 (1:1000, #8242, Cell Signaling Technologies), NLRP3 (1:1000, #15,101, Cell Signaling Technologies), Cleaved-Caspase-1 P20 (1:1000, PA5-99390, Invitrogen), Cleaved-GSDMD (1:1000, #50,928, Cell Signaling Technologies), GSDMD (1:1000, ab225867, Abcam, Cambridge, UK), Caspase1 (1:1000, ab138483, Abcam), ASC (1:1000, #67,824, Cell Signaling Technologies), and β-actin (1:200, ab115777, Abcam).

Techniques: Infection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Western Blot, Immunofluorescence, Comparison

Journal: Functional & Integrative Genomics

Article Title: Huang Qin decoction increases SLC6A4 expression and blocks the NFκB-mediated NLRP3/Caspase1/GSDMD pathway to disrupt colitis-associated carcinogenesis

doi: 10.1007/s10142-024-01334-x

Figure Lengend Snippet: A schematic drawing of HQD in CAC. Activation of SLC6A4 expression by HQD decreased the secretion of serotonin. After serotonin secretion was reduced, the NFκB pathway-induced NLRP3/Caspase1/GSDMD pyroptosis pathway was blocked, which alleviated CAC.

Article Snippet: After being blocked with skim milk for 45 min at room temperature, the PVDF membranes were incubated overnight at 4 °C with primary antibodies against SLC6A4 (1:1000, H00006532-D01P, Novus Biological Inc., Littleton, CO, USA), p-NFκB p65 (1:1000, #3033, Cell Signaling Technologies, Beverly, MA, USA), NFκB p65 (1:1000, #8242, Cell Signaling Technologies), NLRP3 (1:1000, #15,101, Cell Signaling Technologies), Cleaved-Caspase-1 P20 (1:1000, PA5-99390, Invitrogen), Cleaved-GSDMD (1:1000, #50,928, Cell Signaling Technologies), GSDMD (1:1000, ab225867, Abcam, Cambridge, UK), Caspase1 (1:1000, ab138483, Abcam), ASC (1:1000, #67,824, Cell Signaling Technologies), and β-actin (1:200, ab115777, Abcam).

Techniques: Activation Assay, Expressing