Relevant proteins were transiently expressed in HEK-293FT cells, followed by cell lysis in a buffer as described previously24 (link),25 (link). After cell lysis, anti-FLAG-coated magnetic beads (Sigma) or anti-His-tagged agarose beads (Qiagen, Hilden, Germany) were added, followed by further incubation at 4 °C for 2 h. The precipitated proteins were washed in the same lysis buffer and subjected to immunoblotting with anti-FLAG (1:2000; F3165, RRID: AB_259529) (Sigma), anti-XBP1 (1:2000; 619501, RRID: AB_319507) (Biolegend, San Diego, CA, USA) and anti-β-actin (1:4000; #8457, RRID: AB_10950489) (Cell Signaling, Danvers, MA, USA). Cells were also harvested at the indicated time points, and whole-cell-lysates were analyzed by immunoblotting with anti-XBP1 (Biolegend), anti-Fbw7 (1:2000; SC-293423), anti-p50 (NF-κB) (1:2000; SC-8414, RRID: AB_628015), anti-E2F-1 (1:2000; SC-251, RRID: AB_627476) (Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-actin (Cell Signaling), and anti-Lamin B (1:2000; PA 50043) (AB Frontier, Seoul, Korea) antibodies.
Anti xbp1
Manufactured by BioLegend
Sourced in United States
Anti-XBP1s is a laboratory reagent that detects the active, spliced form of the transcription factor XBP1. XBP1 is a key regulator of the unfolded protein response, which is important in cellular stress and homeostasis. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Anti ire1α, by Cell Signaling Technology (5 mentions)
Anti phospho eif2α, by Cell Signaling Technology (3 mentions)
Anti p erk, by Cell Signaling Technology (3 mentions)
Anti flag, by Merck Group (2 mentions)
Anti hsp90, by Santa Cruz Biotechnology (2 mentions)
Anti atf4, by Santa Cruz Biotechnology (2 mentions)
Anti eif2α, by Santa Cruz Biotechnology (2 mentions)
Anti atf6, by Cosmo Bio (2 mentions)
Anti tbp, by Abcam (2 mentions)
Criterion tgx precast sds page gels, by Bio-Rad (2 mentions)
Anti flag m2, by Merck Group (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Anti actin, by Merck Group (2 mentions)
Anti e2f1, by Santa Cruz Biotechnology (1 mentions)
Anti flag coated magnetic beads, by Merck Group (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Lds loading buffer, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
18 protocols using anti xbp1
1
Affinity-based Protein Purification and Analysis
2
Monitoring UPR Pathway Activation
3
Western Blot Analysis of ER Stress Markers
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Immunoblotting was performed as described previously (Nakada et al., 2010 (link)). Briefly, the same number of cells (20,000-30,000) from each test population were sorted into Trichloroacetic acid (TCA) and adjusted to a final concentration of 10% TCA. Extracts were incubated on ice for 15 minutes and spun down for 10 minutes at 13,000 rpm at 4°C. The supernatant was removed and the pellets were washed with acetone twice then dried. The protein pellets were solubilized with Solubilization buffer (9 M Urea, 2% Triton X-100, 1% DTT) before adding LDS loading buffer (Invitrogen, Carlsbad, CA). Proteins were separated on a Bis-Tris polyacrylamide gel (ThermoFisher Scientific, Waltham, MA) and transferred to a PVDF membrane (Millipore, Billerica, MA). Antibodies were anti-Ire1α (Cell Signaling Technology, 3294), anti-Xbp1s (Biolegend, Poly6195), and anti-ß-actin (A1978, Sigma).
4
ChIP-seq antibody usage protocol
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The following antibodies were used for chIP assays: normal rabbit IgG (2729; Cell Signaling Technology) at a 1:200 dilution and anti-XBP1s (619502; BioLegend) at a 1:100 dilution.
5
Immunostaining of Phospho-IRE1 in Hippocampal Neurons
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Primary cultured hippocampal neurons were fixed in 4% paraformaldehyde for 1 h and permeabilized in 0.1% Triton‐X 100 for 10 min, followed by treatment with 1% bovine serum albumin for 20 min. These procedures were performed at room temperature (25°C). The following antibodies were used; anti‐IRE1 (1 : 500; Cell Signaling Technology, RRID: AB_823545), anti‐microtubule‐associated protein 2 (MAP2) (1 : 1000; EMD Millipore, Billerica, MA, USA, RRID: AB_11213363), anti‐MAP2 (1 : 500; Abcam, Cambridge, UK, RRID: AB_2138153), anti‐postsynaptic density protein 95 (PSD95) (1 : 500; NeuroMab, Davis, CA, USA, RRID: AB_2307331), anti‐XBP1s (1 : 500; BioLegend, Sandiego, CA, USA, RRID: AB_2562960), and Anti‐phospho‐IRE1α (1 : 500). Cells were visualized under a FV1000D confocal microscope (Olympus, Tokyo, Japan). P‐IRE1 and MAP2 fluorescence intensities 40–90 μm from somata in randomly selected dendrites were measured, using ImageJ (National Institutes of Health, Rockville, MD, USA). The number of P‐IRE1‐positive puncta overlapping with PSD95‐positive postsynaptic sites was manually counted 40–90 μm from somata in randomly selected dendrites. The cells were randomly chosen from five independent cultures.
6
Transcriptional Regulation Analysis by ChIP Assay
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The PTH1R or IHH luciferase reporter plasmid and XBP1s or negative control plasmid were transfected into 293T cells by using PEI. The activity of firefly and renilla luciferases was quantified using a dual luciferase reporter gene detection system (Promega Corp., Madison, WI, USA). To standardize the difference in transfection, firefly luciferase activity was normalized to that of renilla luciferase.
C28/I2 cells were submerged in DMEM containing 1% formaldehyde and incubated at 37 °C for 10 min. Fixation was stopped by the addition of 0.125 M glycine. The cell lysate was harvested and sheared with 12 pulses of 3% amplitude. Each pulse consisted of a 2.5-s sonication followed by a 7.5-s rest on ice to prevent heat build-up. The sample was incubated with 2 μg of anti-XBP1s (BioLegend, Cat. No. 647502) antibody or IgG (Cat. No. 2729) overnight at 4 °C on a vertical mixer. The enriched DNA fragments were harvested by using methods according to the manufacturer's instructions for the ChIP assay kit (Beyotime, P2078). The IHH (−747 to −553) and PTH1R (−1338 to −1137) promoter regions were amplified. The amount of immunoprecipitated DNA in each sample was represented as a percentage of the total amount of input chromatin, which was equivalent to 100%. The primers used in ChIP are listed inTable S1 .
C28/I2 cells were submerged in DMEM containing 1% formaldehyde and incubated at 37 °C for 10 min. Fixation was stopped by the addition of 0.125 M glycine. The cell lysate was harvested and sheared with 12 pulses of 3% amplitude. Each pulse consisted of a 2.5-s sonication followed by a 7.5-s rest on ice to prevent heat build-up. The sample was incubated with 2 μg of anti-XBP1s (BioLegend, Cat. No. 647502) antibody or IgG (Cat. No. 2729) overnight at 4 °C on a vertical mixer. The enriched DNA fragments were harvested by using methods according to the manufacturer's instructions for the ChIP assay kit (Beyotime, P2078). The IHH (−747 to −553) and PTH1R (−1338 to −1137) promoter regions were amplified. The amount of immunoprecipitated DNA in each sample was represented as a percentage of the total amount of input chromatin, which was equivalent to 100%. The primers used in ChIP are listed in
7
Western Blotting Quantification Techniques
8
ChIP Assay for XBP1s Complexes
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ChIP assays were performed with an Agarose ChIP Kit (Pierce, Cat# 26156, Thermo Fisher Scientific), according to the manufacturer’s instructions. In brief, 293T cells were subjected to cross-linking with 1% formaldehyde, and glycine solution was then added to stop the cross-linking process. Nuclear extracts were prepared. Chromatin-XBP1s complexes were immunoprecipitated with anti-Flag (Sigma, Cat# F3165; diluted 1: 500, St. Louis, MO, USA) or anti-XBP1s (BioLegend, Cat# 647501; diluted 1:100) antibodies by incubation at 4 °C overnight, and this was followed by incubation with beads from the Agarose ChIP Kit (Pierce) or Protein G-Sepharose beads (GE Health, Chicago, IL, USA) at 4 °C for 1 h with gentle rocking. After the beads were washed 5 times with wash buffer, the complexes were eluted from the beads with elution buffer and subjected to PCR analysis.
9
Immunocytochemistry of Neonatal Rat Cardiomyocytes
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For immunocytochemistry (ICC) of NRCs, cells were grown on Lab-Tek II chamber slides (Thermo Scientific, 154461) plated at a density of 1 × 105 cells/well. Subsequently, immunofluorescent microscopy was performed essentially as described previously [48-50 (link)]. Cells were first washed with PBS, fixed for 15 min in 4% paraformaldehyde, permeabilized with 0.5% Triton-X 100 in PBS, and exposed to an antigen retrieval solution (0.1 M glycine, pH 3.5) for 30 min. The fixed cells were incubated in blocking solution (1% BSA, 0.1% cold water fish gelatin, 0.1% Tween-20, 0.05% sodium azide in PBS) for 1 h. Primary antibody incubation was performed overnight at 4°C, whereas the secondary antibody (1:100) along with counterstains were applied for 1 h at room temperature. The following primary antibodies were used: anti-Sigmar1 (1:100, Invitrogen) anti-actin (1:1000, Sigma–Aldrich), anti-TnI (1:1000, Millipore), anti-CHOP (1:100, Cell Signaling, Technologies, Inc.), α-cardiac actin (1:1000, Sigma–Aldrich), anti-XBP1s (1:200, BioLegend). Coverslips were fixed with Vectashield Hardset (Vector Labs, H1400). Nuclei were stained with DAPI (Invitrogen). Cells were imaged using a Leica TCS SP5 Spectral Confocal Microscope loaded with Leica LAS (AF 2.6.3) and NIS-Elements (AR 4.13.04) software.
10
Western Blot Analysis of UPR Markers
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