1 × 106 MDMs were lysed in 100 μL of NETN buffer (100 mM NaCl, 20 mM Tris-Cl pH 8.0, 0.5 mM EDTA, 0.05% NP-40) with protease inhibitors (cOmplete, Roche) and phosphatase inhibitor (PhosphoStop, Roche), and protein concentration was determined using BCA protein assay kit (Pierce). Proteins were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore). Membranes were probed with antibodies to MAVS (Cell Signaling Technology), STING (ProteinTech), vinculin (Cell Signaling Technology), or β-actin (Sigma), developed using Western Lightening Chemiluminescent Substrate (Perkin Elmer), and analyzed in a ChemiDoc imager (Bio-Rad Laboratories).
Western lightening chemiluminescent substrate
Manufactured by PerkinElmer
Western Lightening Chemiluminescent Substrate is a luminol-based chemiluminescent reagent used for the detection of proteins in Western blotting applications. It generates a luminescent signal when exposed to horseradish peroxidase-labeled secondary antibodies, allowing for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (3 mentions)
Vinculin, by Cell Signaling Technology (2 mentions)
Chemidoc imager, by Bio-Rad (2 mentions)
Sting, by Proteintech (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Phosphostop, by Roche (2 mentions)
Spliced xbp1, by BioLegend (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
3 protocols using western lightening chemiluminescent substrate
1
MAVS and STING Protein Detection
2
Quantitative Immunoblotting of Innate Immune Proteins
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1 × 106 MDMs were lysed in 100 μl of NETN buffer (100 mM NaCl, 20 mM Tris–Cl pH 8.0, 0.5 mM EDTA, 0.05% NP‐40) with protease inhibitors (cOmplete; Roche) and phosphatase inhibitor (PhosphoStop; Roche), and protein concentration was determined using BCA protein assay kit (Pierce). Proteins were separated on SDS‐PAGE gels and transferred to PVDF membranes (Millipore). Membranes were probed with antibodies to MAVS (Cell Signaling Technology), STING (ProteinTech), vinculin (Cell Signaling Technology), or β‐actin (Sigma), developed using Western Lightening Chemiluminescent Substrate (PerkinElmer), and analyzed in a ChemiDoc imager (Bio‐Rad Laboratories).
3
Western Blotting for UPR Protein Analysis
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Western blotting procedures have been described previously. The nitrocellulose membranes were blocked with the specific blocking solution for 2 h at room temperature. The nitrocellulose membranes were then treated with specific primary antibodies, including PERK (Cell Signalling, Danvers, MA, USA, Cat #3192), ATF6 (Abcam, Hong Kong, China, Cat #ab122897), Spliced XBP1 (Bio Legend, San Diego, CA, USA, Cat #619501), c-MYC (Santa Cruz Biotechnology Cat # sc-40, Dallas, TX, USA) and β-Actin (Sigma, London, UK, Cat #A-5060) at 4 °C overnight. After washing three times with PBS/0.05%Tween solution, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. The membranes were then washed twice with PBS/0.05%Tween and once with PBS, and finally, the signals were detected using Western Lightening chemiluminescent substrate (PERKin Elmer, Groningen, The Netherlands, Cat #NEL104001EA).
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