Proteins (50 μg) from all samples were processed as previously described [28 (link)]. Membranes were incubated for 12 h at 4 °C with primary antibodies to anti-TLR4 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-MyD88 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NFκB (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NLRP3 (3 µg/mL; Novus, Dallas, TX, USA), anti-Caspase-1(1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-IL-1β (1 µg/mL; Thermo Fisher, Waltham, MA, USA) and β-actin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). Samples were maintained at room temperature for 30 min with peroxidase-conjugated secondary antibody diluted 1:1000 in 1 × Tris Buffered Saline (TBS), 5% milk and 0.05% Tween-20 (Signa-Aldrich, Milan, Italy) [29 (link)]. To visualize protein bands, the Electrochemiluminescence (ECL) method was used, and the protein levels were measured by means of the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA).
Anti myd88
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-MyD88 is a primary antibody that specifically targets the MyD88 protein. MyD88 is an adaptor protein that plays a central role in the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling pathways. This antibody can be used for the detection and study of MyD88 in various applications, such as Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti tlr4, by Santa Cruz Biotechnology (4 mentions)
Anti p65, by Santa Cruz Biotechnology (3 mentions)
Anti il 1β, by Thermo Fisher Scientific (2 mentions)
Anti caspase 1, by Santa Cruz Biotechnology (2 mentions)
Anti nlrp3, by Novus Biologicals (2 mentions)
Anti nf κb, by Santa Cruz Biotechnology (2 mentions)
Anti iκbα, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Histone h3, by Abcam (1 mentions)
Anti socs3, by Abcam (1 mentions)
β actin, by Beyotime (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti nos2, by Santa Cruz Biotechnology (1 mentions)
Primary antibody against gapdh, by Santa Cruz Biotechnology (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Anti phospho iκb, by Cell Signaling Technology (1 mentions)
Peroxidase conjugated bovine anti mouse igg secondary antibody, by Jackson ImmunoResearch (1 mentions)
11 protocols using anti myd88
1
Immunoblotting Analysis of Inflammation Markers
2
Lung Inflammation Signaling Pathway
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Lung tissues samples were obtained as described in the “Materials and Methods ” section “Lung Wet/Dry Ratios.” The nuclear extract and total protein were prepared, and the procedure of western blot analysis was performed as described above. Expressions of NF-κB phospho-p65 and phospho-IRF3 in the nuclear extracts were analyzed by immunoblotting with phospho-specific anti-NF-κB p65 (cat. no. AN371; monoclonal rabbit anti-rat; Beyotime) (1:500 in TBST) antibody or phospho-specific anti-IRF3 (cat. no. ab138449; polyclonal rabbit anti-rat; Abcam, Cambridge, UK) (1:500 in TBST) antibody, and histone H3 (cat. no. sc-374,669; monoclonal mouse anti-rat; Santa Cruz) (1:1000 in TBST) was used as a lysate control. Expressions of TLR4, MyD88, TRIF, and SOCS3 in total protein were analyzed by immunoblotting with anti-TLR4 (cat. no. sc-293,072; monoclonal mouse anti-rat; Santa Cruz) (1:500 in TBST), anti-MyD88 (cat. no. 4283; monoclonal rabbit anti-rat; Cell Signaling Technology) (1:1000 in TBST), rabbit polyclonal anti-TRIF (cat. no. ab13810; polyclonal rabbit anti-rat; Abcam, Cambridge, UK) (1:500 in TBST), or anti-SOCS3 (cat. no. sc-73,045; monoclonal mouse anti-rat; Santa Cruz) (1:200 in TBST). β-actin (Beyotime) (1:1000 in TBST) was used as a lysate control. Corresponding HRP-conjugated secondary antibodies (Beijing Golden Bridge) were used to display the protein signal.
3
Western Blot Analysis of TLR4, MyD88, and NOS2
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Cells were harvested, washed thrice with cold PBS followed by lysis in REPA lysis buffer containing 1% protease inhibitor cocktail (Sigma) and 3% phosphatise inhibitor cocktail (Sigma). Total protein concentration in cell lysate was evaluated using Bradford reagent (Sigma) and lysates containing equal amounts of protein (50 μg/well) were resolved by SDS PAGE and electroblotted to nitrocellulose membrane. Membranes were blocked in 5% w/v skimmed milk in 1X TBST for 30 minutes and incubated with rabbit polyclonal anti-TLR4 (1:1000 v/v), anti-MyD88 (1:1000 v/v), and anti-NOS2 (1:1000 v/v, all from Santa Cruz Biotechnology). Rabbit polyclonal anti–β-actin (1:500 v/v, Cell Signalling Technology), was used as loading control. Membranes were washed with 1xTBST and incubated with Goat anti-rabbit secondary antibody HRP conjugate (1:2000 v/v, polyclonal, Roche) for 2 h and visualized with Enhanced Chemiluminisence kit (Roche) according to manufacturer’s protocol.
4
Western Blot Analysis of Inflammatory Signaling
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A Western blot analysis was performed as already described [43 (link)]. Membranes were probed with the following primary antibodies: anti-phospho- IκB (Cell Signaling 2859), anti-MyD88 (Santa Cruz Biotechnologies, sc-74532), and anti-TLR4 (Santa Cruz Biotechnologies, sc-293072) in 1× PBS, 0.1% Tween-20, 5% w/v non-fat dried milk (PMT) at 4 °C overnight [44 (link),45 (link)]. The membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) [46 (link)]. The blots were also incubated with the primary antibody against GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA). Signals were detected with an enhanced chemiluminescence detection system reagent according to the manufacturer’s instructions (SuperSignalWest Pico Chemiluminescent Substrate, Pierce, WA, USA) [46 (link)].
5
Cellular Trafficking and Lysosomal Modulation
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LPS, mevastatin, and FITC-CTB were purchased from MilliporeSigma. Chloroquine, PSC-833, and 2‑hydroxypropyl-βCD were purchased from GE Healthcare (now Cytiva), Valspodar, and InvivoGen, respectively. PRX consisting of 2-(2-hydroxyethoxy) ethyl carbamate–modified βCDs as cyclic molecules, Pluronic P123 as an axle polymer, and N-triphenylmethyl groups as acid-cleavable stopper molecules were synthesized as described previously (45 (link), 46 (link)). The antibodies used were anti-Flag (catalog F1804; MilliporeSigma), anti-GST (PM013; MBL), anti-CFP (D153-3; MBL), anti-Myd88 (sc-74532; Santa Cruz Biotechnology), anti-p65 (sc-372; Santa Cruz Biotechnology), anti-GAPDH (ab181602; Abcam), anti-LAMP1 (sc-20011; Santa Cruz Biotechnology), anti-Rab7 (9367), anti-EE1A (3288; Cell Signaling Technology), anti-PDI (3501; Cell Signaling Technology), anti-Lamin A/C (4777; Cell Signaling Technology), and anti-CTB (SAB4200844; MilliporeSigma).
6
Immunoprecipitation and Immunoblot Analysis
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Cells were stimulated with 1 μg/ml LPS for the indicated time periods and lysed in weak RIPA buffer (0.5% Triton X-100, 120 mM NaCl, 50 mM Tris-HCl; pH = 7.5) containing phosphatase and protease inhibitors. The whole-cell lysate was sequentially incubated with one of the following antibodies: anti-MyD88, anti-p85α, anti-Nck1, anti-BCAP, or anti-pTyr (Santa Cruz, sc-18182) and shaken overnight at 4°C. Protein A/G-agarose beads (MedChemExpress USA, HY-K0202) were added into the mixture and shaken for a further 2 h, prior to elution with 1 × SDS sample buffer. Prepared samples were further analyzed by immunoblot.
7
Alpha-synuclein-induced TLR2/MyD88 Interaction
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BV-2 cells were treated with 10 µM of wtTIDM or mTIDM. After 30 min of TIDM treatment, fibrillar α-syn (0.5 µM or 7 µg/ml) was added to the cells and incubated for 2 h. Following the incubation period, cells were scraped and lysed in RIPA buffer. The cell homogenate was centrifuged at 17,500 × g for 15-min 4 °C, the supernatant was collected and protein was estimated using the BCA method. The cell lysate was immunoprecipitated with 2 µg of anti-TLR2 or anti-MyD88 or normal IgG (Santa Cruz Biotechnology) overnight at 4 °C, followed by incubation with protein A-agarose for 4 h at 4 °C. Protein A-agarose-antigen-antibody complexes were collected by centrifugation at 10,000 × g for 1 min at 4 °C. The pellets were washed 3–4 times with 1 ml of IP buffer containing 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 10% glycerol, and protease inhibitor cocktails for 20 min each time at 4 °C. Bound proteins were resolved by SDS-PAGE, followed by western blotting with the anti-MyD88 (1:1000, Santa Cruz Biotechnology) and/or anti-TLR2 (1:1000, Abcam). Input from each sample was also run in the western blotting.
8
Immunoblotting of 293T Cells with EXOSC3 and CNOT4
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293T cells with or without forced expression of EXOSC3 and CNOT4 were lysed with lysis buffer (10 mmol/L Tris‐HCl [pH 7.4], 5 mmol/L EDTA, 150 mmol/L NaCl, 10% glycerol, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% SDS, 50 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride [PMSF], and 1 mmol/L sodium orthovanadate [Na3VO4]) and a protease inhibitor mixture (complete [EDTA‐free] protease inhibitor [Roche]) for 20 min on ice and centrifuged at 15,106 g for 15 min at 4°C. Supernatants were subjected to SDS‐PAGE, and the separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore). The membranes were incubated with primary antibodies at 4°C overnight and then incubated with horseradish peroxidase‐labeled secondary antibodies. The signals were developed using ECL™ Western Blotting Detection Reagents (GE Healthcare) and visualized with an LAS 4000 mini system (GE Healthcare). The antibodies used in this experiment were as follows: anti‐FLAG® M2‐peroxidase (HRP) (Sigma), anti‐phospho‐p44/42 MAPK (T202/Y204) (D13.14.4E) XP™, anti‐phospho‐JNK, anti‐phospho‐STAT3 (Y705), anti‐phospho‐AKT (S473) (D9E) XP™ (Cell Signaling Technologies), anti‐MYD88 (Santa Cruz Biotechnology), and anti‐actin (clone C4) (Millipore).
9
Immunoblotting of NF-kB Signaling Pathway
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Total cellular extracts were prepared using 1X Cell Lysis Buffer (Cell Signaling Technology, USA) supplemented with 1 mM PMSF. Protein concentration was determined using the Bradford assay. Cell extracts were subjected to sodium do-decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and gels were transferred to Hybond ECL nitrocellulose membranes (GE Heathcare, Germany) for 100 min at 90 V. The membranes were blocked with 5% skim milk in 1X tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature. The following antibodies were used for protein detection: anti-IκBα, anti-p65, anti-IKKα, anti-IKKβ, anti-MyD88, anti-IRAK1, anti-TRAF6, anti-TAK1, anti-actin antibodies (Santa Cruz Biotechnology Inc.), anti-phosphorylated TAK1 (p-TAK1) at Ser 412, anti-p-IKKα/β at Ser176/180, anti-p-IκBα at Ser32, and anti-p-Akt at Ser473 antibodies (Cell Signaling Technology, USA), and anti-p-IRAK-1 at Thr209 (Abcam, USA). After successful washes, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. Blots were developed using West Pico Western blot detection kit (Thermo Fisher Scientific).
10
Immunofluorescence Analysis of Innate Immune Signaling
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Samples were fixed with 4% paraformaldehyde in 0.1 M of PBS (Lonza, Basel, Switzerland). Then, cells were permeabilized with 0.5% Triton X-100 in PBS (Lonza, Basel, Switzerland) for 10 min and blocked with 5% skimmed milk in PBS for 30 min [27 (link)]. The following primary antibodies were used in the study: anti-TLR4 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-MyD88 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NFκB (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NLRP3(1:500; Novus, Centennial, CO, USA), anti-Caspase-1 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-IL-1β (5 µg/mL; ThermoFisher, Waltham, MA, USA). Cells were incubated with primary antibody for 2 h at room temperature. Then, samples were incubated with Alexa Fluor 568 red fluorescence conjugated goat anti-rabbit secondary antibody (1:200; Molecular Probes, Invitrogen, Eugene, OR, USA) for 1 h at 37 °C. To stain the cytoskeleton actin, cells were treated with Alexa Fluor 488 phalloidin green fluorescent conjugate (1:400, Molecular Probes, Invitrogen, Eugene, OR, USA) for 1 h, and to stain the nuclei, cells were stained with TOPRO (1:200; Molecular Probes, Invitrogen, Eugene, OR, USA) for 1 h. The Zeiss LSM800 confocal system (Zeiss, Jena, Germany) was used to acquire microphotographs.
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