Proteins were prepared as described previously.16 (link) Freeze-clamped LV tissues (200–300 mg) were homogenized briefly in 10 volumes of lysis buffer containing (in mM) 20 Tris-HCl (pH, 7.4), 150 NaCl, 2.5 EDTA, 50 NaF, 0.1 Na4P2O7, 1 Na3VO4, 1 PMSF, 1 DTT, 0.02% (v/v) protease cocktail (Sigma-Aldrich), 1% (v/v) Triton X-100, and 10% (v/v) glycerol. The homogenates were centrifuged two times at 20 000 × g at 4 °C for 15 min, and the supernatants were saved as total proteins. Protein concentrations were determined by the BCA method. Equal amounts of proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). Western blot analysis was performed under standard conditions with specific antibodies, including anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-PKCɛ (Ser729), anti-PKCɛ, anti-phospho-GSK-3β (Ser9), anti-GSK-3β, anti-PERK, anti-Xbp-1s, anti-p50-ATF6 (Abcam, London, UK), anti-TRAF2, anti-GRP78, anti-caspase-12 (Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antibodies were purchased from Cell Signaling (Danvers, MA, USA), unless noted otherwise. The immunoreaction was visualized using an enhanced chemiluminescent detection kit (Amersham, London, UK), exposed to X-ray film, and quantified by densitometry with a video documentation system (Gel Doc 2000; Bio-Rad).
Anti xbp1
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-XBP1 is a laboratory reagent that can be used to detect the expression of the XBP1 protein. XBP1 is a transcription factor that plays a role in the unfolded protein response. The anti-XBP1 reagent can be used in various experimental techniques, such as Western blotting, to measure the levels of XBP1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti grp78, by Abcam (7 mentions)
Tissue culture plate, by Corning (4 mentions)
Cell culture reagents, by Thermo Fisher Scientific (4 mentions)
Anti chop, by Cell Signaling Technology (4 mentions)
Anti atf6, by Abcam (4 mentions)
Anti caspase 12, by Abcam (3 mentions)
Anti grp78 bip, by Abcam (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti ire1α, by Cell Signaling Technology (3 mentions)
Anti atf4, by Abcam (3 mentions)
Anti phospho eif2α, by Cell Signaling Technology (3 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Tunicamycin tum, by Merck Group (2 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
Anti atf6, by Proteintech (2 mentions)
Thapsigargin, by Merck Group (2 mentions)
Anti β actin, by Merck Group (2 mentions)
23 protocols using anti xbp1
1
Western Blot Analysis of LV Proteins
2
Cell Culture Reagents and Antibodies
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Cell culture reagents were purchased from Thermo-Fisher (Carlsbad, CA, USA), and tissue culture plates and other plastic materials were obtained from Corning Inc. (Corning, NY, USA). Salts, buffers, and MTT were obtained from Merck (Darmstadt, Germany). Doxorubicin (99% of purity) was obtained from Merck (Darmstadt, Germany). Antibodies anti-XBP1s and anti-p-eIF4E were purchased from Abcam (Cambridge, UK). Anti-β-actin, anti-eIF4A, anti-PDCD4, anti-c-Jun, anti-MMP-9, anti-Nrf2, anti-Mdm2, anti-Flot-2, anti-CD63, anti-Hsp90, anti-Hsp70, anti-p53, anti-p70S6Kα, and anti-p-p70S6Kα were obtained from Sta. Cruz Biotechnology (Dallas, TX, USA).
3
Molecular Mechanisms of Cellular Responses
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Cell culture reagents were purchased from Thermo-Fisher Scientific (Waltham, MA, USA), tissue culture plates and plastic materials were obtained from Corning (Corning, NY, USA). RPMI-1640 and DMEM media, trypsin, fetal bovine serum (FBS), penicillin, streptomycin, and amphotericin B were obtained from Thermo-Fisher Scientific. MTT, auraptene (Aur > 98% of purity), and tunicamycin (Tum) were obtained from Merck. Anti-SREBP2, anti-HMGCR, anti-LDL-R, anti-β-adaptin, anti-ACE2, anti-HNF1α, anti-AT1R, anti-TMPRSS2, anti-AT1R, anti-PDX-1, anti-Bcl-2, anti-BAX, anti-Lamin B, anti-GAPDH, anti-PMCA1/4, anti-SERCA2, anti-NCX1, anti-CHOP, anti- NF-κB, anti-IL-6, anti-COX-2, Anti-XBP1s, anti-PDI, anti-ABCA1, anti-ABCG, anti-PPARα, anti-ACAT, and anti-β-actin antibodies were obtained from Sta. Cruz Biotechnology (Dallas, TX, USA). Anti-XBP1s was purchased from Abcam (Cambridge, UK).
4
Evaluating Cell Response to Endoplasmic Reticulum Stress
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Cell culture reagents were purchased from Thermo-Fisher (Carlsbad, CA, USA), tissue culture plates and other plastic materials were obtained from Corning Inc. (Corning, NY, USA). Salts and buffers were obtained from Merck (Darmstadt, Germany). Doxorubicin (Dox), MTT, salubrinal (Sal) and tunicamycin (Tum) were obtained from Merck. Antibodies anti-XBP1s and anti-BiP/GRP78 were purchased from Abcam (Cambridge, UK). Anti-β actin, anti-CD47, anti-PCNA, anti-eIF4A, anti-PDCD4, anti-c-Jun, anti-MMP-9, anti-Nfr2, anti-BECN1, anti-Mdm2, anti-PERK, anti-CHOP, anti-FAK, anti-p-FAK were obtained from Sta. Cruz Biotechnology (Dallas, TX, USA).
5
Characterization of Lipid-Based Reagents
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Cell culture reagents were purchased from Thermo Fisher (Carlsbad, CA, USA), while tissue culture plates and other plastic materials were obtained from Corning Inc. (Corning, NY, USA). Salts and buffers were obtained from Sigma-Aldrich (St. Luis, MI, USA), as well as Thioflavin T (ThT), black Sudan B, Congo red, sodium dodecyl sulfate (SDS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). l -α-phosphatidylcholine (PC), l -α-phosphatidylserine (PS), l -α-phosphatidyl-ethanolamine (PE), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (LPA), 1-palmitoyl-2-oleoyl-sn-glycerol (POPG), and cholesterol were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Antibodies anti-XBP1s and anti-BiP/GRP78 were purchased from Abcam (Cambridge, UK) and anti-β-actin was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-PDI was donated by Dr. Marco A. Ramos Ibarra.
6
Western Blot Analysis of UPR Proteins
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The cells were placed in RIPA lysis solution containing PMSF on ice for 30 minutes and the cells were scraped. Protein supernatant was collected after centrifugation at 4°C, 14 000 r/min for 15 minutes. Protein quantification was performed using BCA kits. 4 × Loading buffer was added into the protein supernatant and then heated for 10 minutes at 100°C to prepare protein samples. The proteins were isolated by SDS-PAGE gel electrophoresis and transferred to PVDF membrane. The protein-loaded membranes were immersed in skim milk (5%) at 37°C for 1 hour. Next, it was incubated with specific primary antibodies at 4°C overnight. The antibodies included anti-BMAL1 (1:1000, Abcam), anti-GRP78 (1:500, Biogot Biotechnology), anti-PERK (1:1000, Cell Signaling Technology), anti-IRE1ɑ (1:1000, Abcam), anti-XBP1 (1:500, Abcam), anti-ATF6 (1:1000, Proteintech), anti-ATF4 (1:500, Proteintech), anti-BAX (1:1000, Abcam), anti-BCL-2 (1:1000, Abcam), anti-β-actin (1:1000, Abmart). β-actin was used in WB as the housekeeping protein. The corresponding secondary antibody (1:10 000, Beyotime) was incubated 2 hours at room temperature, and enhanced chemiluminescence reagent (ThermoFisher) was used to detect the protein band signal. Image J software was used for strip analysis and its gray value was calculated.
7
Characterization of CFTR Regulatory Pathways
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Thapsigargin (Sigma-Aldrich, T9033), STF-083010 (TOCRIS, 4509), APY29 (TOCRIS, 4865), MSC 2032964A (TOCRIS, 5641), VX-809 (Selleckchem, Chemical Abstracts Service registry no. 936727-05-8), VX-770 (Selleckchem, CAS registry no. 873054-44-5), and cycloheximide (Sigma-Aldrich, C4859) were purchased commercially. CSTMP was custom-synthesized by Cayman Chemical (Ann Arbor, MI, USA) (CAS registry no. 1000672-89-8).
The following antibodies were acquired commercially: anti-CFTR M3A7 (Millipore, Billerica, MA, USA), anti-IRE1α (Cell Signaling Technology, 3294), anti–phospho-S724 IRE1α (Abcam, ab48187), anti-XBP1 (Abcam, ab198999), anti-ASK1 (Cell Signaling Technology, 3762), anti–phospho-Thr845 ASK1 (Cell Signaling Technology, 3765), anti-BiP (Cell Signaling Technology, 3177), anti-CHOP (Cell Signaling Technology, 2895), anti-pro/p17–caspase 3, anti–cleaved PARP1 (Abcam, ab136812), anti–Na- and K-dependent ATPase (Na,K-ATPase) α1 (Cell Signaling Technology, 23565), anti-HA (Cell Signaling Technology, 2367), anti-Myc (Cell Signaling Technology, 2276), anti–aldolase A (Abcam, ab78339) anti–β-actin (Santa Cruz Biotechnology, sc47778), and anti-pendrin (Santa Cruz Biotechnology, sc23779). The anti-R4 polyclonal antibody was raised against peptides corresponding to amino acids 1458 to 1471 of human CFTR, as described previously (12 (link)).
The following antibodies were acquired commercially: anti-CFTR M3A7 (Millipore, Billerica, MA, USA), anti-IRE1α (Cell Signaling Technology, 3294), anti–phospho-S724 IRE1α (Abcam, ab48187), anti-XBP1 (Abcam, ab198999), anti-ASK1 (Cell Signaling Technology, 3762), anti–phospho-Thr845 ASK1 (Cell Signaling Technology, 3765), anti-BiP (Cell Signaling Technology, 3177), anti-CHOP (Cell Signaling Technology, 2895), anti-pro/p17–caspase 3, anti–cleaved PARP1 (Abcam, ab136812), anti–Na- and K-dependent ATPase (Na,K-ATPase) α1 (Cell Signaling Technology, 23565), anti-HA (Cell Signaling Technology, 2367), anti-Myc (Cell Signaling Technology, 2276), anti–aldolase A (Abcam, ab78339) anti–β-actin (Santa Cruz Biotechnology, sc47778), and anti-pendrin (Santa Cruz Biotechnology, sc23779). The anti-R4 polyclonal antibody was raised against peptides corresponding to amino acids 1458 to 1471 of human CFTR, as described previously (12 (link)).
8
Western Blot Analysis of Protein Expression
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Protein expression was detected by Western blot analysis as previously described [30 (link)]. The following antibodies were used: anti-β-actin (1:10000) was obtained from Sigma-Aldrich (#A5441, St. Louis, MO, USA); anti-GRP78 (1:5000), anti-phosphorylated EIF2α (1: 2000), anti-EIF2α (1:2000), anti-XBP1 (1:5000), anti-caspase12 (1:5000) and anti-ATF4 (1:2000) were obtained from Abcam Trading [Shanghai] Company Ltd. (#ab21685, #ab32157, #ab169528, #ab37152, #ab62463, #ab1371, respectively); anti-ATF6 (1:3000) was obtained from ABclonal Biotechnology, Boston, USA (#A2570); anti-P-Erk1/2 (1:2000), anti-Erk1/2 (1:2000), anti-caspase3 (1:5000), anti-Bcl-2 (1:2000) and anti-Bax (1:2000) were obtained from Cell Signaling Technology, Danvers, MA, USA (#4370, #4695, #14220, #3498, #2772, respectively); anti-CHOP (1:1000) was obtained from GeneTex, Irvine, USA (#GTX32616); anti-Mfrn1 (1:2000) was obtained from Proteintech, Wuhan, China (#26469-1-AP). The immunoreactive proteins were detected using the enhanced chemiluminescence (ECL) method and quantified by transmittance densitometry using ImageJ software.
9
Western Blot Analysis of UPR Markers
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Western blotting analysis follows the standard procedures as previously described [25 (link)]. Primary antibodies using as follows: anti-GRP78 (1:1000, Abcam, USA), anti-XBP1 (1:500, Abcam, USA), anti-CHOP (1:500, Cell Signaling Technology, Inc, USA) and anti-Cx32 (1:1000, Sigma-Aldrich, USA). Then, secondary antibody (Millipore, USA) and anti-GAPDH (Cell Signaling Technology, Inc, USA) were used at 1:5000. Image J scanning software was used to scan the image and the data were described as relative values to Sham values.
10
Immunohistochemical Analysis of ER Stress Markers
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Renal tissue slides were de-waxed with xylene and dewatered with ethanol, then were microwave-heated to antigen retrieval in citrate buffer for 30 min. Nonspecific sites were blocked in goat serum. Next, sections incubated with anti-GRP78 (1:1000, Abcam, USA), anti-XBP1 (1:200, Abcam, USA), anti-CHOP (1:3000, Cell Signaling Technology, Inc, USA & 1:100, Abcam, USA) at 4 °C overnight, and then incubated with secondary antibody (1:500, Life technologies, USA) at 37 °C for 30 min. Ten fields of each section were chosen randomly to observe positively stained areas [26 (link)].
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