The total proteins in the MDBK cells were treated with the serum-free medium. BVDV and BVDV + MT were extracted 36 h post-infection using the BCA Protein Assay Kit (Thermo Fisher Scientific Co. Ltd., Shanghai, China) according to the manufacturer’s guidelines. The extracted proteins were separated by reducing SDS-PAGE electrophoresis and transferred onto a PVDF membrane blocked with 5% nonfat milk in Tris-Tween-buffered saline buffer for 1.5 h. The membranes were then incubated with primary antibodies [IκB, p-IκB, p65, and p-p65 (all 1:1000 from Abmart, Shanghai, China) and E2 (1:1000)] and secondary antibodies [Goat anti-mouse IgG (1:5000, SA00001-1; ProteinTech Group, Rosemont, IL, USA) and Goat anti-rabbit IgG (1:5000, SA00001-2; ProteinTech Group, USA)] conjugated with horseradish peroxidase. The gray values for each band were quantified using ImageJ software and normalized to those of GAPDH (1:5000, 10494-1-AP; ProteinTech Group, USA). E2 antibody was generated in rabbits and produced by our laboratory.
P iκb
Manufactured by Abmart
Sourced in China
P-IκB is a laboratory product used for protein analysis. It is a phosphorylated form of the IκB protein, which plays a role in the regulation of the NF-κB signaling pathway. P-IκB can be used in various biochemical and cell-based assays to study the activation of the NF-κB pathway.
Automatically generated - may contain errors
Lab products found in correlation
Nf κb p65, by Abmart (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg, by Proteintech (1 mentions)
Sa00001 1, by Proteintech (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Goat anti mouse igg, by Proteintech (1 mentions)
P nf κb p65, by Wanlei (1 mentions)
Cy3 goat anti rabbit igg h l, by ABclonal (1 mentions)
Atf 4, by Wanlei (1 mentions)
Fitc goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Hrp goat anti rabbit igg, by ABclonal (1 mentions)
P62 sqstm1, by Proteintech (1 mentions)
Hydroxychloroquine hcq, by Merck Group (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Beclin1, by ABclonal (1 mentions)
β actin, by ABclonal (1 mentions)
P erk, by Wanlei (1 mentions)
Bcl2 associated x (bax), by ABclonal (1 mentions)
Bcl 2, by ABclonal (1 mentions)
4 protocols using p iκb
1
Western Blot Analysis of BVDV Infection
2
Chalcone, Autophagy, and Cell Signaling
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(E)2’-hydroxychalcone (2’-HC; purity ≥ 98%) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Hydroxychloroquine (HCQ; purity ≥ 98%), 3-Methyladenine (3-MA; purity ≥ 98%), and N-acetylcysteine (NAC; purity ≥ 99%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin (Rap; purity ≥ 98%) was purchased from Aladdin (Shanghai, China).
The primary antibodies used were as follows: β-actin, Bax, Bcl-2, PARP, cleaved-PARP p25, caspase-9, caspase-3, LC3B, Beclin1, and p-IκB. They were purchased from ABclonal (Wuhan, China). p62/SQSTM1 was obtained from Proteintech (Wuhan, China). ERK, JNK, p-JNK, p38, p-p38, NF-κBp65, IκB, p-IκB, p-eIF2α, and MMP9 were obtained from Abmart (Shanghai, China). P-ERK, p-NF-κBp65, ATF-4, and CHOP were purchased from Wanleibio (Shenyang, China). HRP Goat Anti-Rabbit IgG (Abclonal), Alexa Flour 594-Goat Anti-Rabbit IgG (Abbox, Jiangsu, China), Cy3 Goat Anti-Rabbit IgG (H + L) (Abclonal), and FITC Goat Anti-Rabbit IgG (Servicebio, Wuhan, China) were used as secondary antibodies for a Western blot or immunofluorescence.
The primary antibodies used were as follows: β-actin, Bax, Bcl-2, PARP, cleaved-PARP p25, caspase-9, caspase-3, LC3B, Beclin1, and p-IκB. They were purchased from ABclonal (Wuhan, China). p62/SQSTM1 was obtained from Proteintech (Wuhan, China). ERK, JNK, p-JNK, p38, p-p38, NF-κBp65, IκB, p-IκB, p-eIF2α, and MMP9 were obtained from Abmart (Shanghai, China). P-ERK, p-NF-κBp65, ATF-4, and CHOP were purchased from Wanleibio (Shenyang, China). HRP Goat Anti-Rabbit IgG (Abclonal), Alexa Flour 594-Goat Anti-Rabbit IgG (Abbox, Jiangsu, China), Cy3 Goat Anti-Rabbit IgG (H + L) (Abclonal), and FITC Goat Anti-Rabbit IgG (Servicebio, Wuhan, China) were used as secondary antibodies for a Western blot or immunofluorescence.
3
Immunoblotting of Inflammatory Markers
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The protein lysates were separated on SDS-PAGE gels and then transferred to PVDF membranes (Invitrogen, Waltham, United States). The membranes were blocked in 5% BSA for 1 h and incubated with following primary antibodies: TNF-α (1:1,000, Santa Cruz, California, United States), P65, IκB, and p-IκB (1:1,000, Abmart, Shanghai, China), E-cadherin (1:1,000, Affinity, Nanjing, China), SMAD4 (1:1,000, Cell Signaling Technology, Danvers, United States), α-SMA (1:1,000, Merck, Darmstadt, Germany), FN (1:1,000, Abcam, Cambridge, United Kingdom), TGF-βRII, SMAD2/3, p-SMAD2/3 (1:1,000, Elabscience, Wuhan, China), and β-actin (1:1,000, Santa Cruz, California, United States) overnight at 4°C. The membranes were then washed three times with TBST for 10 min and incubated with secondary antibody (1:5,000, ZSGB-BIO, Beijing, China). The protein bands were visualized using ECL reagents.
4
Western Blot Analysis of Inflammasome Signaling
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RIMVECs were seeded, divided into groups, and treated as described in the in vitro experiment paragraph. Total proteins were extracted using the radio immunoprecipitation assay (RIPA) cell lysis buffer for 10 min, and protein concentration was determined using the bicinchoninic acid (BCA) protein detection kit (Beyotime Biotechnology, China). A standard western blot protocol was performed, and the following primary antibodies were used overnight at 4°C: TLR4 (1:800) (ABclonal, Wuhan, China), NLRP3 (1:1000) (ABclonal), GSDMD (1:1000) (ABclonal), caspase-1 (1:1000) (ABclonal), ASC (1:1000) (ABclonal), NF-κB p65 (1:1000) (Abmart, Shanghai, China), p-NF-κB p65 (1:400) (Abmart), p-I-κB (1:1000) (Abmart), I-κB (1:1000) (Abmart), IL-18 (1:1000) (Affinity Biosciences, Colorado, USA), IL-1β (1:1000) (Affinity Biosciences), claudin-1 (1:1000) (Affinity Biosciences), claudin-2 (1:1000) (Affinity Biosciences), ZO-1 (1:1000) (Affinity Biosciences), and β-tubulin (1:1000) (Affinity Biosciences) used as loading control, followed by the incubation with HRP (Horseradish Peroxidase)-conjugated secondary antibodies (1:5000) (ABclonal) for 1 h at room temperature. The blots were visualized using a Tanon 5200 chemiluminescence imaging system (Shanghai, China) and quantified using the ImageJ software (National Institutes of Mental Health, Bethesda, MD, USA).
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