After rutin and TEGDMA treatment, the cells were lysed by radioimmunoprecipitation (RIPA) lysis buffer containing protease inhibitors and phosphatase inhibitors as in previous studies [57 (link)]. Cellular proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked and then blotted with primary antibodies against HO-1, Nrf-2, phosphorylation of AMPK, AMPK, β-actin (Santa Cruz Biotech, Dallas, TX, USA) overnight at 4 °C. The membranes were washed and further incubated with secondary antibodies. Finally, the membranes were imaged by enhanced chemiluminescence kit using the Fusion Solo S (Vilber Lourmat, Collégien, France). The band intensities of protein expression and phosphorylation were quantified using Evolution Capt software (Vilber Lourmat Fusion Solo S). The levels of protein expression and phosphorylation were expressed relative to endogenous control levels.
Fusion solo s
Manufactured by Vilber
Sourced in France, Germany, United States, Japan
The Fusion Solo S is a laboratory equipment product designed for DNA and protein gel imaging. It features a high-resolution camera and specific lighting for accurate detection of fluorescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Nacalai Tesque (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
β actin, by Merck Group (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Blocking one, by Nacalai Tesque (3 mentions)
Supersignal west dura substrate, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Differential quik stain kit, by Sysmex (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Western lightning plus ecl, by PerkinElmer (2 mentions)
Ab125096, by Abcam (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Mcl 1, by Cell Signaling Technology (2 mentions)
Ab18251, by Abcam (2 mentions)
Supersignal chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Chemi lumi one, by Nacalai Tesque (2 mentions)
Bca kit, by Beyotime (2 mentions)
90 protocols using fusion solo s
1
Western Blot Analysis of Antioxidant Pathways
2
Western Blot Analysis of Cellular Markers
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Samples corresponding to 0.1–0.4 OD600 units of cells were separated by SDS–PAGE followed by Western blotting and immunodecoration with primary antibodies raised against mCherry (1:2,000, ab125096; Abcam), Pgk1 (1:10,000, ab113687; Abcam), GFP (1:1,000, 13921700; Roche), HA (1:5,000, A2095; Sigma-Aldrich), and Atg11 (1:1,000, gift from Dr. Hayashi Yamamoto, Nippon Medical School). After treatment with the secondary antibodies, HRP-conjugated rabbit anti-mouse IgG (H + L) for mCherry, GFP, HA, and Pgk1, and goat anti-rabbit mouse IgG (H + L) for Atg11 (1:10,000, 315-035-003 and 111-035-003, respectively; Jackson ImmunoResearch) followed by the enhanced chemiluminescence reagent Western Lightning Plus-ECL (203-19151; PerkinElmer) or ImmunoStar LD (PTJ2005; Wako), proteins were detected using a luminescent image analyzer (FUSION Solo S; VILBER). Quantification of the signals was performed using FUSION Solo S (VILBER).
3
Quantitative Western Blot Analysis
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Samples corresponding to 0.1–0.4 OD600 units of cells were separated by SDS-PAGE followed by western blotting and immunodecoration with primary antibodies raised against mCherry (1:2000, Abcam ab125096), Pgk1 (1:10,000, Abcam, ab113687), HA (1:5000, 16B12, Covance), Ape1 (1:5000), Atg8 (1:2000) (gifts from Dr. Hitoshi Nakatogawa, Tokyo Institute of Technology, Japan), and Atg11 (1:1000) (a gift from Dr. Hayashi Yamamoto, Nippon Medical School, Japan). After treatment with the secondary antibodies, horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (H + L) for mCherry, HA, Pgk1, and goat anti-rabbit IgG (H + L) for Ape1, Atg8 and Atg11 (1:10,000, Jackson ImmunoResearch 315-035-003 and 111-035-003, respectively) followed by the enhanced chemiluminescence reagent Western Lightning Plus-ECL (PerkinElmer, 203-19151) or Chemi-Lumi One L (nacalai tesque, 07880-70), proteins were detected using a luminescent image analyzer (FUSION Solo S; VILBER). Quantification of the signals was performed using FUSION Solo S (VILBER).
4
Western Blot Analysis of Protein Signaling
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Total cellular protein was isolated from the cells after various treatments. For Western blots, a previously described procedure was applied [54 (link)]. The following primary antibodies were used: Acetyl Histone H3, HDAC1, HDAC8, PPARγ, cyclin D1, CDK6, p-Ser473 Akt, Akt, p-Ser2448 mTOR, mTOR, p-Ser139 H2AX, H2AX, Bax, Mcl-1, PARP, procaspase-8, cleaved caspase-9, LC3B, and Atg5 were purchased from Cell Signaling Technologies (Beverly, MA, USA); β-actin, Sigma-Aldrich (St. Louis, MO, USA). The secondary antibodies were purchased from Santa Cruz Biotechnology. The enhanced chemiluminescence (ECL) system for detection of immunoblotted proteins was from GE Healthcare Bioscience (Piscataway, NJ, USA). Then, the protein was visualized by FUSION SOLO S (VILBER, Deutschland, Germany).
5
Cell Lysis and Western Blotting
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The preparation of cell lysates and western blotting were performed as described previously (75 (link)). The primary antibodies used were anti-GAS41 (sc-393708, Santa Cruz), anti-H2A.Z (ab4174, Abcam; 2718, CST), and anti-α-tubulin (ab18251, Abcam). Luminescence was analyzed on a FUSION SOLO S (Vilber).
6
Western Blot Analysis of Signaling Pathways
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Cells seeded in 25 cm2 culture bottles were lysed with cold RIPA Lysate reagent (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. The protein concentrations were measured with the BCA assay kit. Proteins (40 µg) were separated by 12% SDS-PAGE and transferred to 0.45 µm PVDF membranes (Merck Millipore, Darmstadt, Hesse-Darmstadt, Germany). After the membranes were blocked, they were incubated overnight at 4 °C with the diluted primary antibodies against the following molecules: phosphorylated Smad1/5/9, Smad1, phosphorylated PKA, PKA, phosphorylated CREB, CREB and GAPDH. Then, the membranes were washed with TBST and incubated with diluted secondary antibodies for 1 h at room temperature. Finally, we visualized the membranes with an enhanced ECL substrate kit (Millipore, Billerica, MA, USA) on the Fusion SOLO S (Vilber, Collégien, France). The densities of the product bands were quantified using ImageJ software and standardized to that of GAPDH.
7
Monitoring HER3 Regulation by IgG 3-43
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Example 6
Cellular HER3 expression levels and IgG localization after incubation of IgG 3-43 were analyzed by western blot and immuno-fluorescence microscopy, respectively. For Western blot analysis, MCF-7 cells were seeded in 6 well plates two days before, to be semi confluent on the day of the experiment. Cells were serum starved for one night and incubated with 100 nM IgG 3-43 for the indicated time points. HER3 levels were analyzed by western blot. Density of the bands was analyzed with the Fusion Solo S software (Vilber). The values were corrected for loading differences by reactivation to tubulin loading control and normalized to the relative values of untreated probes. IgG 3-43 rapidly leads to a reduction of HER3 levels in MCF-7 cells (
8
Quantitative Image and Western Blot Analysis
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Hypocotyl and root lengths and GUS intensity (mean gray value) were measured and quantified with the ImageJ (http://rsb.info.nih.gov/ij /) software. The root meristem length, width, and the fluorescence signal intensity (mean gray value) of the presented markers was quantified on raw images using the Leica software (LAS AF Lite). The western blot signals were quantified using a Fusion Solo S (Vilber).
Means and standard errors were calculated and the statistical significance was evaluated using the Graph Pad Prism5 (http://www.graphpad.com ) software. The significance of the data was evaluated using the Student’s t test in the case of two columns comparisons. One-way ANOVA followed by Tukey’s test was performed in the case of the multiple columns’ comparisons procedure. Two-way ANOVA followed by Bonferroni post-tests was carried out to compare two different genotypes at different treatments.
Representative data are shown throughout the text. All experiments have been performed in at least three replications.
Means and standard errors were calculated and the statistical significance was evaluated using the Graph Pad Prism5 (
Representative data are shown throughout the text. All experiments have been performed in at least three replications.
9
Western Blot Analysis of Neural Stem Cells
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NSCs were washed once with PBS(-) and lysed using RIPA buffer (Nacalai Tesque Inc.) with a protease inhibitor cocktail (#08714, Nacalai Tesque Inc.). We performed sodium dodecyl sulfate‒polyacrylamide gel electrophoresis using 4–12% Bolt™ Bis-Tris and a 1.0 mm Mini Protein Gel system. The proteins were transferred onto a polyvinylidene fluoride membrane (#1214726, GVS, S.p.A. Rome, Italy) and analyzed by western blotting. The primary and secondary antibodies used are listed in Supplementary Table 1 . The luminescent signal was obtained using a horseradish peroxidase substrate (Amersham™ ECL™ Prime, Cytiva, Tokyo, Japan) and captured using Fusion Solo-S (Vilber, Collégien, France). As the internal control, we detected beta-Actin protein using the same membrane reprobed after treatment with stripping solution (Fuji Film Wako Chemical Inc.).
10
Western Blot Analysis of SDF1 Protein
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The lysis of NPCs was performed for total protein with a RIPA lysis buffer (Beyotime Institute of Biotechnology). The concentrations of every group were tested using a BCA protein quantitation kit (Beyotime Institute of Biotechnology). Proteins from cell lysates were separated via 12% SDS-PAGE (Beyotime Institute of Biotechnology) and 40 µg protein was loaded per lane, then transferred to polyvinylidene fluoride membranes (EMD Millipore), which were blocked using 5% skimmed milk for 1 h at room temperature. The membranes were incubated at 4°C overnight with primary antibodies against SDF1 (1:1,000; ab9797, Abcam) and GAPDH (1:3,000; 10494-1-AP, Proteintech Group, Inc.). After being washed in a TBST solution, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1,000; A0208, Beyotime Institute of Biotechnology) for 1 h at 37°C. The protein bands were detected using an ECL kit (Wanleibio Co., Ltd.) and a chemiluminescence imaging system (Fusion SOLO S, VILBER). Protein expression was quantified using EvolutionCapt SL6 software (v16.0.8.0, Vilber Lourmat Sté).
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