Western blotting assays were used to identify the different expressions of protein. Modified RIPA lysis buffer was used to collect the total cell lysates. BCA Protein Assay Kit (Beyotime, Jiangsu, China) was used to standardize the concentration of different samples. Proteins were separated by SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane by using Mini-PROTEAN® Tetra (Bio-Rad, Hercules, CA, USA), and probed with primary antibodies respectively. The primary antibodies were as follows: YAP (D8H1X) XP® Rabbit mAb (1:1000, 14074S, Cell signaling technology), SOX2 Polyclonal antibody (1:1000, 11064-1-AP; Proteintech), OCT4 Polyclonal antibody (1:1000, 11263-1-AP; Proteintech), NANOG Polyclonal antibody (1:1000, 14295-1-AP; Proteintech), CD44 Polyclonal antibody (1:2000, 15675-1-AP; Proteintech), CD133 Polyclonal antibody (1:2000, 18470-1-AP; Proteintech), GAPDH Monoclonal antibody (1:50000, 60004-1-lg; Proteintech), Alpha Tubulin Polyclonal antibody (1:2000, 11224-1-AP; Proteintech). Subsequently the membranes were incubated with corresponding secondary antibodies: HRP-Goat anti-rabbit IgG (H+L) (RS0002; Immunoway); HRP-Goat anti-mouse IgG (H+L) (RS0001; Immunoway). Blots were detected using an enhanced chemiluminescence system (S6, manufactured by CLiNX, Shanghai, China).
Hrp goat anti rabbit igg h l
Manufactured by Immunoway
Sourced in United States
HRP-Goat anti-rabbit IgG (H+L) is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to rabbit immunoglobulin G (IgG) antibodies in a variety of immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Hrp goat anti mouse igg h l, by Immunoway (2 mentions)
Gapdh monoclonal antibody, by Proteintech (1 mentions)
Yap d8h1x xp rabbit mab, by Cell Signaling Technology (1 mentions)
Alpha tubulin, by Proteintech (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Mini protean tetra, by Bio-Rad (1 mentions)
Transintro el transfection reagent, by Transgene (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Chemiluminescent hrp substrate, by Merck Group (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Caspase 1, by Immunoway (1 mentions)
β actin, by Immunoway (1 mentions)
5 protocols using hrp goat anti rabbit igg h l
1
Protein Expression Analysis by Western Blotting
2
Plasmid Transfection and NS1 Expression
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DEFs were seeded in a 6-well plate (2 × 106/well) and cultured at 37℃ with 5% CO2. The pVAX1, pVAX1-NS1, pVAX1-TPA-NS1, and pVAX1-EC30-NS1 plasmids were separately transfected into DEFs when the cells reached 90% confluence, using TransIntroTM EL Transfection Reagent (TransGen Biotech) according to the manufacturer's instructions. The cell supernatants and pellets were collected at 48 h post transfection. The cell pellets were lysed in 100 μL radio immunoprecipitation assay (RIPA ) lysis Buffer (Beyotime, Shanghai, China) containing 1% phenylmethanesulfonyl fluoride (PMSF , Beyotime, Shanghai, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE ) in a 15% acrylamide gel was performed to resolve the cell supernatants and lysates. Then the proteins were transferred onto a PVDF membrane (Bio-rad; CA), 220 mA, 80 min. The membrane was blocked with 5% nonfat milk for 2 h at room temperature. Rabbit anti-NS1 polyclonal antibody (prepared in this study) with 1:1,000 dilution and horseradish peroxidase (HRP )-goat anti-rabbit IgG(H+L) (Immunoway, Plano, TX) with 1:5,000 dilution were used to analyze the NS1 protein expression. The Clarity Western ECL Substrate (Bio-Rad, CA) was used for the membrane imaging.
3
Protein Expression Analysis in Liver Cells
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HepG2, SMMC-7721 cells and collected tissues were assayed using RIPA (radio-immunoprecipitation assay) buffer. BCA was used to measure total protein concentration. Proteins were then separated by 10% SDS-PAGE and transferred to PVDF (provided by Bio-Rad, Hercules, USA) membranes. The membranes were blocked with 5% nonfat milk and incubated with mono-antibodies against POLA2 (Absin, abs138967, 70KDa), GAPDH (Proteintech, 1E6D9, 36 KDa) and PD-L1 (Huabio, ET1701-41, 33 KDa). Afterward, the membranes were washed with TBST and incubated with secondary antibodies HRP* Goat Anti Mouse IgG (H + L) ( Immunoway, RS0001, China) or HRP* Goat Anti-Rabbit IgG (H + L) (RS0002, Immunoway, China). The results were measured with an enhanced chemiluminescence system. The imageQuant LAS500 was provided by GE Health Care (Fairfield, America). Chemiluminescent HRP Substrate was obtained from Millipore Corporation (Billerica, America). The intensity of the protein bands was determined by ImageJ software. GAPDH protein was used for normalization.
4
Western Blot Analysis of Caspase-1
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This section was performed in accordance with the former studies. Briefly, proteins from liver tissues were extracted by sonication in RIPA lysis buffer with phenylmethanesulfonylfluoride. After centrifugation at 12,000 rpm for 10 min at 4°C, the supernatant of the lysate was collected and the protein concentration was detected by the BCA method. The proteins of samples were fractionated through 8% SDS-PAGE and transferred onto NC membranes. After being blocked with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with different primary antibodies overnight at 4°C. After washing with TBST three times (10 min each), the blots were incubated with secondary antibodies for 1 h. Finally, the signal was visualized using ECL reagent. Pro-Caspase-1 (#YT0652, 1:1,000), Caspase-1 (#YC0002, 1:1,000), β-actin (#YT0099, 1:5,000), and secondary antibody HRP* Goat Anti Rabbit IgG(H+L) (#RS0002, 1:10,000) antibody were purchased from ImmunoWay Biotechnology (Plano, USA). Western blot quantification was analyzed densitometrically using ImageJ software and normalized to β-actin.
5
cGAS-STING Signaling Pathway Activation
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Cells were first lysed using RIPA Lysis Buffer (containing 1% protease and phosphatase inhibitors). Centrifuge at 13000 g for 15 min at 4 °C, collect protein samples from the supernatant, and perform protein quantification by BCA method. The target protein and the carrier protein buffer are mixed according to the ratio of 3. After boiling for 10 minutes, an equal amount of sample was loaded on 10% SDS-PAGE separation gel and electrophoresed (S1 80 V for 20 mins, S2 120 V 90 mins). Next, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (constant current 150 mA, 150mins) and blocked with rapid blocking solution for 30mins at room temperature according to the reagent manufacturer’s instructions. Membranes were then incubated with primary antibody (cGAS, STING, pSTING, Actin) overnight at 4 °C, followed by secondary antibody incubation for 60 mins. Finally, ECL system was used for exposure and color development, and ImageJ was used for image processing. Primary or secondary antibodies, including cGAS (D3O8O) Rabbit mAb (CST, #31659), STING (D2P2F) Rabbit mAb (CST, #13647), Phospho-STING (Ser365) (D8F4W) Rabbit mAb (CST, #72971) and HRP* Goat Anti Rabbit IgG(H+L), ImmunoWay Biotechnology Company (TX,75024 USA), #RS0002, were used.
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