Western blotting analysis was carried out as previously described [25 (link)]. The following antibodies were used: anti-Bcl-2 (#ab182858), anti-Bax (#ab182733), anti-Bcl-xl (#ab32370), anti-p-P65 (#ab76302), anti-P65 (#ab32536), anti-p-IκBα (#ab133462), anti-IκBα (#ab32518) (Abcam Cambridge, MA, USA), anti-FXR (#sc-25309), anti-TLR4 (#sc-293072) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-ERK1/2 (#4370), anti-ERK1/2 (#4695), anti-p-P38 (#4511), anti-P38 (#8690), anti-p-JNK1/2 (#4668), anti-JNK1/2 (#9252), anti-CCL2 (#66272-1-Ig), and anti-β-actin (#66009-1-Ig) (Proteintech, Wuhan, China).
Anti fxr
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-FXR is a laboratory product designed to target the Farnesoid X receptor (FXR), a nuclear receptor that plays a crucial role in bile acid, lipid, and glucose metabolism. This product is intended for use in research applications, and its core function is to modulate the activity of FXR for experimental purposes.
Automatically generated - may contain errors
Lab products found in correlation
Anti cyp7a1, by Santa Cruz Biotechnology (2 mentions)
Anti bcl xl, by Abcam (1 mentions)
Anti iκbα, by Abcam (1 mentions)
Anti p p65, by Abcam (1 mentions)
Anti p iκbα, by Abcam (1 mentions)
Anti p erk1 2, by Proteintech (1 mentions)
Anti p p38, by Proteintech (1 mentions)
Anti p38, by Proteintech (1 mentions)
Anti p jnk1 2, by Proteintech (1 mentions)
Anti jnk1 2, by Proteintech (1 mentions)
Anti ccl2, by Proteintech (1 mentions)
Anti tlr4, by Santa Cruz Biotechnology (1 mentions)
Anti p65, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti erk1 2, by Proteintech (1 mentions)
Dehydrocholic acid, by Merck Group (1 mentions)
Gcdca, by Merck Group (1 mentions)
Tcdca, by Merck Group (1 mentions)
6 protocols using anti fxr
1
Western Blotting Analysis of Apoptosis and Inflammation Signaling
2
Inflammatory Bowel Disease Biomarker Analysis
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OEA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); DSS (molecular weight=36,000–50,000 kDa) was obtained from MP Biochemical (Santa Ana, CA). MPA O-glucuronide and naloxone 3-β-D-glucuronide were obtained from Toronto Research Chemicals Inc. (ON, Canada). β-MCA, UDCA, HDCA, G-LCA, G-UDCA, G-DCA, T-β-MCA, T-UDCA, T-HDCA and T-LCA were purchased from Steraloids Inc. (Newport, RI). α-MCA, CA, DCA, CDCA, lithocholic acid (LCA), G-CDCA, G-CA, T-CDCA, T-DCA, T-CA, dehydrocholic acid and propranolol were purchased from Sigma-Aldrich (St Louis, MO). The anti-UGT1A, anti-FXR, anti-FGF15 and anti-CYP7A1 antibodies were purchased from Santa Cruz Biotechnology. The anti-PPARα antibody was from Abcam (Cambridge, UK). The anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from SunShine Biotechnology (Nanjing, China). The secondary antibodies and recombinant human FGF19 was obtained from Bioworld Technology (St Louis Park, MN). Other reagents, unless mentioned, were obtained from Sigma-Aldrich. RNA extracts of colon biopsies from 8 healthy humans and 13 ulcerative colitis and Crohn’s disease patients were previously described51 (link). The ulcerative colitis and Crohn’s disease samples were collected from consented patients and approved by the University of Michigan IRB committee (approval number HUM00042210).
3
Western Blot Analysis of Liver Proteins
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Frozen liver tissues were lysed in a RIPA buffer supplemented with cOmplete™ Mini Protease Inhibitor Cocktail (Roche) and phosphatase inhibitors (PhosSTOP EASYpack; Roche). Extracted proteins (60 μg) were resolved in SDS polyacrylamide gels, and then transferred onto a PVDF membrane using semi-dry transfer (Immobilon-P; Millipore). The following primary antibodies were used: anti-FGF19 (MAB969, R&D Systems; ab154185, Abcam), anti-FGFR4 (8562, Cell Signalling), anti-CYP7A1 (sc-25536, Santa Cruz), anti-CYP3A4 (sc-53850, Santa Cruz), anti-SHP (sc-15283, Santa Cruz), anti-FXR (sc-1204, Santa Cruz), anti-CAR (PP-N4111-00, R&D), and anti-OSTβ (sc-163192, Santa Cruz). Protein loading was normalized to GAPDH (sc-25778 + HRP; Santa Cruz), β-actin (sc-47778, Santa Cruz), and α/β-tubulin (#2148, Cell Signaling). The bands were visualized using the SuperSignal West Pico Chemiluminescent Detection System (Thermo Scientific). Image and densitometry analyses were performed using MicroChemi Imaging Systems and GelQuant software (DNR Bio-Imaging, Israel).
4
Western Blot Analysis of TGR5, FXR, EGFR and ERK1/2
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MKN45 and MKN74 cells were lysed and proteins (50 μg) were separated by 10% SDS- polyacrylamide gels, followed by electro-transfer onto a nitrocellulose membrane. For EGFR and Erk1/2 western blotting only MKN45 cells were lysed and proteins (50 μg) were separated by 8% and 12% SDS-polyacrylamide gels respectively, followed by electro-transfer onto a nitrocellulose membrane. The membranes were sequentially incubated with blocking buffer (TBS-Tween containing 5% nonfat dry milk or 5% BSA respectively) for 1 hour at room temperature, and then overnight at 4°C with one of the following antibodies: anti-TGR5 (1:2000, #ab72608 Abcam), anti-FXR (1:2000 #sc-13063 Santa Cruz Biotechnology), anti-EGFR (1:1000, #2232 Cell Signaling), and anti-phospho-EGFR antibody (1:1000, #4407 Cell Signaling), anti-Erk1/2 (P44/42 MAPK) (1:1000, #46955 Cell Signaling), anti-phospho-Erk1/2 (p-P44/42 MAPK) (1:1000, #91015 Cell Signaling). Primary antibody were detected with the horseradish peroxidase (HRP)-labeled secondary antibodies (1:10000, BioRad) for 1 hour at room temperature. After washing with TBST, protein bands were visualized by Lite Ablot TURBO (Euroclone) according to the manufacturer's instructions.
5
Analyzing Transcriptional Regulation of miR-1
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GES-1 cells were transiently transfected with a miR-1 promoter vector. Then, ChIP analysis was performed according to the standard method of the Magna ChIP G Assay kit (#16–662, Millipore, USA). Chromatin was immunoprecipitated with anti-FXR (#SC-25309, Santa Cruz, USA), anti-SNAI2 (#9585, CST, USA) or a negative control IgG. Finally, immunoprecipitated DNA-protein complexes were isolated and a real-time PCR assay was carried out. The primers for the SNAI2 and miR-1 promoters are listed in Table 1 .
6
Immunoblotting Assay for Protein Analysis
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Cells were extracted in RIPA buffer, and immunoprecipitation with specific antibodies was performed using protein A agarose. The pellets were washed 3 times. Immunoblotting was then performed with diluted antibodies overnight at 4°C or for 2 h at room temperature using a standard procedure [16 (link),17 ]. The primary antibodies included: anti-FXR (#sc-25309, 1: 750, Santa Cruz), anti-CREB (#9192, 1: 1000, Cell Signaling Technology), anti-proBDNF (#sc-65514, 1: 750, Santa Cruz), anti-Bax (#ab182733, 1: 500, Abcam), anti-Bcl-2 (#ab32124, 1: 500, Abcam), and anti-β-actin (#A1978, 1: 1000, Sigma-Aldrich). To confirm reproducibility, experiments were performed at least 3 times. Band intensity quantification was carried out using Image J software.
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