Western blot experiments were performed as described by Tan et al. (20 (link)). Primary antibodies used in this study were against SHP (OM107591, Omnimabs, USA), FLAG (14793, CST, USA), phosphor-AMPKα (Thr172) (2531, CST, USA), phosphor-ACC (Ser79) (3661, CST, USA), NRF2 (ab62352, Abcam, USA), H3 (4499, CST, USA) and GAPDH (309154, ZSGB-Bio, China). The immunoreactive protein was visualized using an electrochemiluminescence kit (Beyotime, China) and scanned by an Epson Perfection V33 scanner.
Perfection v33
Manufactured by Epson
Sourced in China
The Epson Perfection V33 is a flatbed scanner designed for personal and home office use. It features a maximum optical resolution of 4800 dpi and can scan documents, photos, and other media up to 8.5 x 11.7 inches in size.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Pall Corporation (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Electrochemiluminescence kit, by Beyotime (1 mentions)
Ab62352, by Abcam (1 mentions)
Gapdh, by ZSGB-BIO (1 mentions)
Imperial protein stain, by Thermo Fisher Scientific (1 mentions)
Irs 1, by Cell Signaling Technology (1 mentions)
Protein extraction kit, by Applygen (1 mentions)
Crystal violet, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Srebp 1c, by Santa Cruz Biotechnology (1 mentions)
Bca method, by Merck Group (1 mentions)
Lamin a c, by Santa Cruz Biotechnology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Pall Corporation (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
C jun, by Cell Signaling Technology (1 mentions)
Total protein extraction kit, by Applygen (1 mentions)
9 protocols using perfection v33
1
Western Blot Analysis of Cellular Targets
2
Basal Phenotypic Analysis of Transgenic Lines
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For the basal phenotypic analysis of transgenic lines and the WT under sufficient nutrient condition, 8-day-old (days after germination) seedlings were transferred to soil. The maximum rosette radius and the plant height were recorded every 3 days, the bolting and flowering times were recorded every day from Stage5.1 and Stage6.0 [53 (link)], respectively. The leaf number at bolting was counted when the inflorescence emergence at the Stage5.1, and the total leaf number was counted after the rosettes growth completed at about Stage6.0. Total number of siliques was counted when the plants growth to Stage6.9, the total seeds produced by a single-plant were harvested at the growth Stage9.7, and the yield per plant was expressed as the weight of total seeds per plant, and the thousand-grain weight were weighed. For N starvation treatments, 7-day-old seedlings of transgenic lines and WT were transferred to NS or ND for 9 days. The phenotypic changes were recorded by scanner (EPSON Perfection v33), the fresh weight of leaves was measured.
3
Flatbed Scanning of Biological Samples
4
Modulating Tomato Root Growth
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For root growth experiments, five-day-old tomato seedlings were incubated in the nutrient solution containing 25 μM CdCl2 or CdCl2 plus various concentrations of NAA (2.5–50 nM), IAA (2.5–50 nM), kyn (2.5–20 μM), yucasin (2.5–20 μM), TIBA (0.5–4 μM), or NPA (1–8 μM) for six days. The solution was refreshed every 2 days. Tomato seedlings were photographed using a scanner (Epson Perfection V33) at 0, 2, 4, and 6 days after treatment, and the root and hypocotyl lengths were measured using the ImageJ software package. IAA (Sangon) was dissolved in 95% ethanol, and NAA (Sangon), NPA (Sigma), TIBA (Sangon), kyn (Macklin), and yucasin (Aladdin) were dissolved in DMSO.
5
SDS-PAGE Gel Staining and Scanning
6
Protein Expression Analysis in Liver Tissue
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Total protein of liver tissue was extracted with Protein Extraction Kit (Applygen Technologies Inc). HepG2 cell lysates were prepared using lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% Triton X-100, protease and phosphatase inhibitor, 0.1 mM PMSF, and 1 μg/mL leupeptin. Harvested lysates were centrifuged at 10,000 ×g for 10 min at 4°C. 50 μg protein was subjected to 10% SDS-PAGE and then transferred to the PVDF membranes (Pall Corporation, Pensacola). We used primary antibodies: Nrf2, SOD2, CAT, GSK3β (pSer9), GSK3β, beta-actin (Santa Cruz Biotechnology), IRS-1 (pSer307), IRS-1, PI3K (pTyr458/199), PI3K, AKT (pSer473), AKT, JNK (pThr183/Tyr), JNK, HO-1 (Cell Signaling Technology), and Nrf2 (pS40) (Abcam). We used secondary antibodies: anti-rabbit antibody, anti-mouse antibody, and anti-goat antibody. Immunoblotting was detected with enhanced chemiluminescence (Pierce Biotechnology, USA). Densities of bands were determined with scanner (Epson Perfection V33).
7
Cell Proliferation Assay with Crystal Violet
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Cells were seeded into six-well plates (1×104 cells/ well) and allowed to grow for 5 days in complete medium. They were then fixed in 10% formalin for 30 minutes at RT and stained for 30 minutes in crystal violet (Fisher Chemical, C581-100) diluted to 1% by volume in H2O. Following H2O washes, samples were dried overnight and imaged on an Epson Perfection V33 scanner.
8
Western Blot Analysis of Liver Proteins
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Whole and nuclear proteins were extracted from liver and HepG2 cells as described previously [10 (link)]. Protein concentration was quantified with the BCA method according to the supplier's instructions (Sigma). 50 μg of protein was added to 8% SDS-PAGE and electroblotted onto PVDF membranes (Pall Corporation, Pensacola). The membranes were blocked and incubated with specific antibodies against AMPK, AMPK (pThr172), FAS, ACC (pSer79), and ACC (cell signaling technology) and SREBP-1c, β-actin, and Lamin A/C (Santa Cruz Biotechnology). Consequently, the membranes were incubated with the corresponding horseradish peroxidase goat anti-rabbit IgG-HRP or anti-mouse IgG-HRP as secondary antibody (Santa Cruz Biotechnology). The immunoreactive proteins were detected with enhanced chemiluminescence (Pierce Biotechnology, USA). Band intensities were scanned (Epson Perfection V33) and quantified using the Image J software.
9
Protein Extraction and Western Blotting
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Total protein was extracted from the liver of experimental fish with Total Protein Extraction Kit (Applygen Technologies Inc, Beijing, China), and nuclear and cytosolic fractions were collected using a nuclear protein extraction kit (Pierce, Nashville, TN, USA), according to the instructions of the manufacturers. Protein concentration was measured using BCA kit (Pierce). Fifty micrograms of protein was loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, USA). The membranes were incubated with 5% skimmed milk for 1 h at room temperature. Immunoblots were obtained using antibodies against the following proteins: ERK1/2, ERK1/2 (pThr202/Tyr204), JNK1/2, JNK1/2(pThr183/Thr185), p38, p38 (pThr180/Thr182), IKKα/β, IKKα/β (pSer176/Ser180), IκBα, IκBα (pSer32/Ser36), c-Jun, c-Jun (pSer73), NF-κB p65 and beta-actin (Cell Signaling Technology, Danvers, MA, USA). Western blots were exposed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA) and film images were scanned by Epson Perfection V33 (China).
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