Antibodies used for immunoblotting, IP, or immunofluorescence were as follows: anti-TAF7 (TAF7 monoclonal antibody M01, clone 2C5, Abnova); anti-TBP (ab63766, Abcam); anti–β-tubulin (ab6046, Abcam); anti-HA (16B12, ab130275, Abcam); anti-FLAG (clone M2, F3165, MilliporeSigma); anti-puromycin (clone 12D10, MABE343, MilliporeSigma); anti-RPL5 (ab86863, Abcam); anti-RPL8 (ab169538, Abcam); anti-XPO1 (M01180, Boster); anti-eIF5B (ab89016, Abcam); anti-hnRNP U (ab20666, Abcam); anti-Haspin (ab21686, Abcam); anti-SMARCC2 (A301-039A, Bethyl); and anti-DPP9 (ab42080, Abcam). The specificity of the anti-TAF7 antibody for TAF7 has been shown previously using MEFs deleted of TAF7 (13 (link)).
Ab42080
Manufactured by Abcam
Sourced in United Kingdom
Ab42080 is a primary antibody that targets the Amyloid Beta (1-42) protein. It is used for research purposes in the study of Alzheimer's disease and other neurodegenerative conditions.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2 agarose resin, by Merck Group (2 mentions)
Sc 47778, by SCBT (2 mentions)
To 12 gradient gels, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Ab63766, by Abcam (1 mentions)
Ab130275, by Abcam (1 mentions)
Ab86863, by Abcam (1 mentions)
Ab169538, by Abcam (1 mentions)
Ab89016, by Abcam (1 mentions)
Ab20666, by Abcam (1 mentions)
Ab21686, by Abcam (1 mentions)
Ab6046, by Abcam (1 mentions)
Ab45167, by Abcam (1 mentions)
Ab124805, by Abcam (1 mentions)
Ab32372, by Abcam (1 mentions)
Ab1416, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Bsa method, by Beyotime (1 mentions)
Anti p53, by Abcam (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
7 protocols using ab42080
1
Antibodies for Immunoblotting, IP, and IF
2
Western Blot Analysis of Protein Markers
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Briefly, cells were lysed in RIPA lysis buffer (P0013B, Beyotime Institute of Biotechnology, Nantong, China) at 48 hr following transfection as described below and protein concentration was determined by the BSA method (Beyotime Institute of Biotechnology). Equivalent quantities of protein were separated on a 10% SDS‐polyacrylamide gels and then transferred to Polyvinylidene Fluoride (PVDF) Membrane. Membranes were blocked using 5% non‐fat milk and incubated overnight with the appropriate primary antibody. The next day, they were washed three times with TBST and incubated with a HRP‐conjugated secondary antibody (Beyotime Institute of Biotechnology) at 1:5,000 dilution for 1 hr at room temperature. Protein detection was performed using the enhanced chemiluminescence (ECL) system (Millipore, Bedford, MA). Primary immunoblotting antibodies were: anti‐β‐Actin(dilution 1:1,000, 4,970, Cell Signaling Technology, Danvers, MA), anti‐DPP9(dilution 1:1,000, ab42080, Abcam, Cambridge, MA), anti‐p53 (Epitomics, Burlingame, CA), anti‐BAX(dilution 1:1,000, ab32503, Abcam), anti‐APAF1(dilution 1:1,000, ab32372, Abcam), anti‐MUC1(dilution 1:1,000, ab45167, Abcam), anti‐S100A4(dilution 1:1,000, ab124805. Abcam), anti‐E‐caderin(1:50, ab1416), anti‐vimentin (1:1,000, ab92547, Abcam).
3
NLRP1 Mutation Immunoprecipitation Protocol
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A total of 2.5 × 105 HEK293T cells were transfected with 500 ng of WT or mutant NLRP1 (IRES-GFP). Eighteen hours posttransfection, cells were washed once with 1×DPBS and harvested in NP40 lysis buffer (1% NP40 (vol/vol), 10% glycerol (vol/vol), 20 nM Tris-HCl, 150 mM NaCl, 1 mM ethyleneglycol-bis-(β-aminoethylether)-N,N,N',N'-tetraacetic acid, 10 mM NaPPi, 5 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride) freshly supplemented with 1× cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland). After lysing cells for 20 minutes on ice, cell debris was spun down and the supernatant was collected. Immunoprecipitation was performed using anti–FLAG-M2-agarose resin (Sigma) for 4 hours or overnight at 4°C. Beads were washed 3 times with lysis buffer before elution by boiling in SDS sample buffer for 10 minutes. Immunoblots were prepared using 4% to 12% gradient gels (Novex, Invitrogen, Carlsbad, Calif) and subsequently transferred to a PVDF membrane. Membranes were blocked in PBS/tween 20 with 5% skim milk for 60 minutes at room temperature and probed overnight at 4°C. The following antibodies were used: aNLRP1: AL176 (AdipoGen, San Diego, Calif), aDPP9: ab42080 (Abcam, Cambridge, UK), aFLAG: 9H1 (in-house), and aActin: sc47778 (SCBT).
4
Western Blot Analysis of Inflammasome Proteins
5
Antibody Detection Protocol for Inflammasome
6
Immunofluorescence Analysis of Inflammasome Proteins
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HaCaT cells or 1° KC were grown on 10 mm2 coverslips at a density of 70’000 cells/coverslip in 24 wells plates. Cells were fixed in 4% paraformaldehyde (PFA) in PBS for 10 min at RT, permeabilized with 0.25% of Triton X-100 in PBS for 10 min and then blocked in PBS containing 1% BSA at room temperature for 30 min as previously described [44 (link)]. Coverslips were incubated with primary antibody ASC (Cat. NBP1-78977 Novus Biological, Littleton, CO, USA) 1:100, NLRP1 (sc-166368 Santa Cruz Biotechnology Inc., Dallas, TX, USA) 1:50, 4HNE (ab46545, Abcam, Cambridge, UK) 1:400, DPP9 (ab42080, Abcam, Cambridge, UK)1:400, UBR2 (18852-1-AP, Proteintech Group Inc., Rosemont, IL, USA) 1: 150 in 0.25% BSA/PBS overnight at 4°C. The Alexa Fluor Fluorochrome-conjugated secondary antibodies (A11004 Alexa Fluor 568, A11008 Alexa Fluor 488 Invitrogen, ThermoFisher Scientific, USA) were used at a dilution of 1:1000 in PBS-BSA 0.25% for 1 h at RT. Nuclei were stained with DAPI (D1306 Invitrogen ThermoFisher Scientific, Waltham, MA, USA) after removal of secondary antibody. Coverslips were mounted onto glass slides using PermaFluor Aqueous Mounting Medium (TA-006-FM, ThermoFisher Scientific, Waltham, MA, USA), and examined using a Zeiss Z1 AxioObserver LSM10 confocal microscope equipped at 40x and 60x magnification. Images were quantified using ImageJ software.
7
Characterization of NLRP1 Mutant Interactions
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About 2.5 × 105 HEK293T cells were plated overnight before cotransfection with 500 ng of wt or mutant pCIG2-hNLRP1-3xFLAG-IRES-eGFP (S1213A or M1184V/S1213A) and 500 ng of empty vector (pcDNA3.1) or Ub-NLRP1-CT-HA. About 18 h post-transfection, cells were washed once with 1× Dulbecco’s PBS and harvested in NP-40 lysis buffer (1% NP-40 [v/v], 10% glycerol [v/v], 20 nM Tris–HCl, 150 mM NaCl, 1 mM EGTA, 10 mM NaPPi, 5 mM NaF, 1 mM Na3VO4, and 1 mM PMSF) freshly supplemented with 1× cOmplete protease inhibitor cocktail (Roche). After lysing cells for 20 min on ice, cell debris was removed by centrifugation, and the supernatant was collected. IP was performed using anti-FLAG-M2-agarose resin (Sigma) for 4 h or overnight at 4 °C. Beads were washed three times with lysis buffer before elution by boiling in SDS sample buffer for 10 min. Immunoblots were prepared using 4 to 12% gradient gels (Novex, Invitrogen) and subsequently transferred to a polyvinylidene difluoride membrane. Membranes were blocked in PBS/Tween-20 with 5% skim milk for 60 min at room temperature and probed overnight at 4 °C. Antibodies: α-NLRP1: AL176 (AdipoGen), α-DPP9: ab42080 (Abcam), α-FLAG: 9H1 (in house), and α-actin: sc47778 (SCBT).
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