Nuclear and cytoplasmic proteins were extracted from HK-2 cells using a Nuclear and Cytoplasmic Protein Extraction Kit (Invitrogen). Proteins (50 µg/lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4°C with anti-ABCA1, -PPARα, -LXRα, -MCP1, -ICAM1, -TGFβ1, -β-actin, and -histone H3 antibodies. Next, the membranes were incubated with goat anti-rabbit or mouse IgG horseradish peroxidase-conjugated secondary antibodies, and then scanned using an Odyssey Fc System (LI/-COR; Amersham, Piscataway, NJ, USA). The intensity of the bands was measured using LabWorks 4.5 software (UVP, Upland, CA, USA).
Labworks 4
Manufactured by Analytik Jena
Sourced in United States, United Kingdom
Labworks 4.0 is a software suite developed by Analytik Jena for the management and analysis of laboratory data. The software provides a centralized platform for data acquisition, storage, and processing from various analytical instruments.
Automatically generated - may contain errors
Lab products found in correlation
Hybond ecl, by Cytiva (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Gapdh, by Analytik Jena (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Biochemi imaging system, by Analytik Jena (2 mentions)
P iκb, by Santa Cruz Biotechnology (2 mentions)
Uvp biochemi imaging system, by Analytik Jena (2 mentions)
Anti p p38, by Cell Signaling Technology (2 mentions)
Anti erk, by Cell Signaling Technology (2 mentions)
Anti gfap, by Abcam (2 mentions)
Anti p jnk, by Cell Signaling Technology (2 mentions)
Anti mao b, by Santa Cruz Biotechnology (2 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
Anti cox 2, by Cell Signaling Technology (2 mentions)
Anti iba1, by Abcam (2 mentions)
Anti p38, by Cell Signaling Technology (2 mentions)
Anti jnk, by Cell Signaling Technology (2 mentions)
Odyssey fc system, by LI COR (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Thermo Fisher Scientific (1 mentions)
34 protocols using labworks 4
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Kidney Proteins
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Whole kidneys were homogenized in ice-cold radioimmunoprecipitation assay lysis buffer, then centrifuged at 14,000× g for 25 minutes at 4°C prior to collection of the supernatants. The protein concentration was measured by Bradford’s method, and the supernatants were stored at −80°C. The cell lysates (50 µg of protein/lane) or whole HK-2 cell extracts (40 µg of protein/lane) were loaded, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to polyvinylidenedifluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4°C with primary antibodies for CTGF (Abcam, Cambridge, UK), TGF-β1, collagen type I (Col1), α-SMA, TIMP-1, PAI-1, E-cad, or β-actin (Santa Cruz Biotechnology). Subsequently, the membranes were incubated with goat antirabbit IgG or goat antimouse IgG HRP conjugate, and then immersed in ECL Plus Western Blotting Detection Reagent (Amersham, Piscataway, NJ, USA) and exposed to Hyperfilm ECL (Amersham). The intensity of the bands was measured using Lab Works 4.5 software (UVP, Upland, CA, USA).
3
Deglycosylation of Purified PRRSV
4
Quantitative Western Blot Analysis of Podocyte Proteins
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Total protein was extracted from podocytes and quantified using the Coomassie brilliant blue method. Eighty-microgram protein samples were denatured in 100°C water for 3 min and rapidly centrifuged for 15 s. A 10% SDS polyacrylamide gel was fixed in an electrophoresis tank, and protein samples were slowly loaded into the wells. Gel electrophoresis was completed at a constant voltage of 90 V. Proteins were transferred to a PVDF membrane by electrophoresis in low-temperature conditions. The PVDF membrane was blocked in TBST with 5% fat-free milk for 2 h. The diluted gremlin antibody (1: 300) and β-actin antibody (1: 1000) were added and incubated with the membrane at 4°C overnight. The PVDF membrane was washed 3 times for 10 min each time. Then, a secondary antibody, IgG (rabbit, 1: 1000) tagged with horseradish peroxidase, was added and incubated with the membrane at 4°C overnight. The bands on the PVDF membrane were visualized using the Odyssey FC imaging system. The Western blot bands were quantitatively analyzed with Lab Works 4.5 software (UVP, US). Results are expressed as the ratio of the optical density of the target and β-actin bands.
5
Western Blot Analysis of Liver Allograft HSCs
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Liver allograft samples were rapidly ground in liquid nitrogen, and the cultured HSCs were harvested. The tissue powder or the isolated HSC cells were reconstituted in ice-cold RIPA buffer with protease and phosphatase inhibitors. Supernatants were recovered after centrifugation and assayed for protein content using the BCA method (Beyotime Biotechnology, P0010). Protein samples of 50 mg per lane were separated by 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene-difluoride (PVDF) membranes. The membranes were incubated in 5% skimmed milk for 2 h at 37°C and overnight at 4°C with primary antibodies (Santa Cruz) (rat anti-CXCR3, 1 : 200 dilution; EGFR, 1 : 200 dilution; JAK2, 1 : 200 dilution; STAT3, 1 : 200 dilution; Collagen IV, 1 : 200 dilution). GAPDH protein expression was used as a loading control. The membranes were exposed to the negative films to develop target bands after incubation with enhanced chemiluminescence (Santa Cruz, USA). The intensities of bands were quantitated by LabWorks 4.5 software (UVP, USA).
6
Western Blot Analysis of Fibrosis Markers
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Total protein was extracted using radioimmunoprecipitation buffer, separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Nanjing Kangruipu Biological Technology Co., Ltd., Nanjing, China) for immunoblotting. The membranes were blocked with 5% defatted milk powder (diluted in TBST) for 1 h and then incubated with rabbit anti-rat FN polyclonal antibody (1:500, Wuhan Boster Biological Engineering Co., Ltd., Wuhan, China) or goat anti-rat Smad2, Smad3, or phosphorylated Smad2/3 (p-Smad2/3) polyclonal antibody (1:500, Abcam, Cambridge, UK) at 4°C overnight. After three washes with PBST, membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (1:5000, Protein Tech Group Inc., Chicago, USA) or rabbit anti-goat IgG (1:9000, Protein Tech Group Inc., Chicago, USA) for 1 h at room temperature. Specific protein bands on the membranes were visualized using enhanced chemiluminescence (Amersham, Piscataway, NJ, USA) according to the manufacturer's instructions, and their absorbance was analyzed using LabWorks 4.5 software (manufacturer: UVP, Upland, USA) and normalized to the β-actin absorbance value.
7
Quantitative RT-PCR Analysis of Trx and Txnip
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Total RNA was extracted from Neuro 2A and Neuro 2A-Trx/LacZ cells in TRIZOL (Takara) according to the manufacturer's instructions. The cDNA was synthesized from 500ng of RNA in a 10μl reaction with RNA PCR Kit (AMV) Ver.3.0 (Takara). GAPDH was used as a housekeeping loading control. The primers used were: Trx (human) forward 5'-GGAAGATCTATGGTGAAGCAGATCGAG-3'，reverse 5'-ACGCGTCGACTTA GACTAATTCATTAATGG-3', LacZ forward 5'-GGAAGATCTGTCGTTTTACAACGTCGT-3', reverse 5'-ACGCGTCGACTTTTTGACACCAGACCAAC-3', Txnip forward 5'-ACTCCTCAAGATGGGTGGCAATC-3', reverse 5'-ACATCCACCCAGCAAACACTCCT-3', GAPDH forward 5'-TGTGATGGGTGTGAACCACGAGAA-3' reverse 5'-GAGCCCTTCCACAATGCCAAAGTT-3'. PCR amplification conditions were as follows: 94°C for 2minutes, 30 cycles of: 94°C for 30 seconds, 55°C for 30 seconds, and 68°C for 3 minutes, and a final extension 72°C for 10minutes. The PCR products were run on a 1% agarose gel at 120 V for 30 minutes and stained with ethidium bromide (EB). The data was analyzed with a GDS-8000 Bio imaging system (UVP, Upland, CA) and Lab Works 4.5 software (UVP).
8
Quantification of Membrane-Bound CPR Mutants
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Expression of the full-length membrane bound CPR mutants was obtained in BTC bacteria and membrane fractions of the different strains were prepared and characterized for protein content as described previously (Marohnic et al., 2010 (link); Palma et al., 2013 (link)). CPR content of membrane fractions was quantified by immunodetection against a standard curve of purified human, full-length WT CPR, using polyclonal rabbit anti-CPR primary antibody and biotin-goat anti-rabbit antibody in combination with the fluorescent streptavidin conjugate (WesternDot 625 Western Blot Kit; Invitrogen). Densitometry of CPR signals was performed using LabWorks 4.6 software (UVP, Cambridge, United Kingdom).
9
Temporal Expression of Connexin Proteins in Hippocampus after Status Epilepticus
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The rats were sacrificed at 5 time points (1 day before SE, and 1, 7, 14, and 28 days after SE). Western blot was used for the semi-quantitative analysis of CX26 (Sigma-Aldrich, United States; diluted 1:400 dilution), CX32 (Sigma-Aldrich, United States; 1:400), CX36 (Sigma-Aldrich, United States; 1:600), and CX43 (Sigma-Aldrich, United States; 1:8000) expression in the hippocampus. Immunoreactive bands were visualized by the ECL Advance Western blot reagent (Bio-Rad, United States). The optical densities of the immunoreactive bands were quantified by densitometry using Labworks 4.6 software (EC3 Imaging System, UVP Inc., United States). The relative levels of the CX26, CX32, CX36, and CX43 were expressed as ratios (CX26/β-actin, CX32/β-actin, CX36/β-actin, and CX43/β-actin).
10
Western Blot Analysis of MAPK Signaling
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Cell lysates were harvested and protein concentrations were determined via bicinchoninic acid protein quantification method [24 (link)]. 40 μg of total protein were separated by SDS-PAGE gel electrophoresis and electrotransferred onto PVDF membranes. Primary rabbit antibodies against total ERK (No. 4695), p-ERK (No. 4370), total AKT (No. 4685), p-AKT (No. 4060), KRAS (No. 3339) (Cell Signaling Technology Inc., Berkeley, CA, USA) and GAPDH (No. TA-08) (loading control) (Zhongshanjinqiao Biotech, China) were incubated at 4 °C overnight at a dilution of 1:1000, and after washed with PBST for three times, the secondary horseradish-peroxidase-labeled antibody was incubated at room temperature for 2 h at a dilution of 1:5000. Finally, the relevant protein was visualized by staining with the enhanced chemiluminescent (ECL) kit (Haigene, Harbin, China). The relative levels of each target protein to the control (total AKT, total ERK, or GAPDH) were determined using a UVP bioimaging system and LabWorks 4.6 software (UVP, Upland, CA, USA).
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