Cell lines were homogenized in Cell Lysis Buffer (Cell Signaling Technology). We measured the protein concentration using a Bio‐Rad protein assay (Bio‐Rad) and added sample buffer solution with reducing reagent (6×) for SDS‐PAGE (Nacalai Tesque) to each sample after matching the concentration. Proteins were separated by SDS‐PAGE and transferred to nitrocellulose membranes. After blocking with blocking buffer (Nacalai Tesque), membranes were incubated with primary Abs overnight at 4°C. The next day, membranes were incubated with HRP‐conjugated secondary Abs (1:5000 anti‐rabbit, 7074; 1:5000 anti‐mouse, 7076; both Cell Signaling Technology). The immobilized peroxidase activity was detected using SuperSignal West Pico PLUS (Thermo Fisher Scientific). The primary Abs used in this study were obtained from the indicated suppliers as follows: rabbit anti‐p‐c‐Jun (1:500, #3270; Cell Signaling Technology), rabbit anti‐c‐Jun (1:1000, #9165; Cell Signaling Technology), and mouse anti‐β‐actin (1:10,000, A1978; Sigma‐Aldrich).
Sds page
Manufactured by Nacalai Tesque
Sourced in Japan
SDS-PAGE is a laboratory technique used to separate proteins based on their molecular weight. It involves the use of sodium dodecyl sulfate (SDS) to denature proteins and an electric field to drive the proteins through a polyacrylamide gel.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary ab, by Cell Signaling Technology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Blocking buffer, by Nacalai Tesque (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Rabbit anti c jun, by Cell Signaling Technology (1 mentions)
Rabbit anti p c jun, by Cell Signaling Technology (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Sds page, by Merck Group (1 mentions)
Immobilon p polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
3 protocols using sds page
1
Western Blot Analysis of Protein Phosphorylation
2
Quantitative analysis of dsRNA
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BHK/T7-9 cells were infected with the WT or T3D-L(S2/3′KRmem) virus, and total RNA was purified from the supernatant and cells using TRIzol LS reagent (Thermo Fisher Scientific). The RNA sample was mixed with an equal volume of the sample buffer solution with 2-mercaptoethanol (2×) for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Nacalai Tesque, Kyoto, Japan) and electrophoresed on a 10% gel at 30 mA for 130 min in an ice bath. dsRNAs were visualized using SYBR Gold nucleic acid gel staining (Thermo Fisher Scientific).
3
Western Blot Analysis of Virus-Infected Cells
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The virus-infected BHK/T7-9 cells were lysed with lysis buffer (1% (v/v) NP-40) and mixed with an equal volume of the sample buffer for SDS-PAGE (Nacalai Tesque). The heat-denatured sample was subjected to SDS-PAGE in a 10% gel, and the separated proteins were electrotransferred to an Immobilon-P polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Burlington, MA, USA). The membrane was blocked with phosphate-buffered saline containing 5% (w/v) skim milk and 0.1% (v/v) Tween-20, and it was incubated with mouse anti-σ3 mAb (clone 4F2, Developmental Studies Hybridoma Bank), mouse anti-β-actin mAb (clone G043, Applied Biological Materials, Vancouver, BC, Canada), or rabbit anti-KillerRed polyclonal antibody (Evrogen, Moscow, Russia). The membrane was then treated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and the chemiluminescent signal was detected using LuminoGraph I (ATTO, Tokyo, Japan).
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