The HaCaT cells and HDF cells were plated at the same density as described above, and the cell lysates were transferred to a new e-tube using PBS after treating with Hs-WE and hydrangenol for 24 h. Then, the cells were centrifuged at 3000 rpm for 3 min. The cell lysates were reacted with cell lysis buffer containing 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 7.5, 20 mM NaF, 2% NP-40, and 50 mM β-glycerol phosphate with 2 µg/mL of pepstatin, leupeptin, and aprotinin. The mixtures were then centrifuged at 12,000 rpm for 10 min after the reaction. The supernatants were used for Western blotting and separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA). The samples were transferred to a PVDF membrane (Merck Millipore) and blocked with a bovine serum albumin (BSA) 3% (w/v) solution. The membrane was incubated overnight with primary antibodies (1:2500 dilution) at 4 °C and was washed for 3 min three times with a TBST solution. Then, it was incubated for 2 h with secondary antibodies at room temperature. The protein bands were detected with enhanced chemiluminescence reagents.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
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Sourced in United States, Italy
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a laboratory technique used for the separation and analysis of proteins based on their molecular weight. It involves the use of a polyacrylamide gel matrix and an electric current to separate proteins according to their size.
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Lab products found in correlation
Bicinchoninic acid assay, by Thermo Fisher Scientific (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (7 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Polyvinylidene fluoride membrane, by Merck Group (6 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (6 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (5 mentions)
Protease inhibitor cocktail, by Roche (5 mentions)
Chemidoc imaging system, by Bio-Rad (5 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
γ tubulin, by Merck Group (4 mentions)
Sonicator, by Qsonica (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Synaptophysin, by Abcam (3 mentions)
Psd95, by Abcam (3 mentions)
Protein assay kit, by Bio-Rad (3 mentions)
Enhanced chemiluminescence reagent, by Merck Group (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
84 protocols using sodium dodecyl sulfate polyacrylamide gel electrophoresis
1
Western Blot Analysis of Hs-WE and Hydrangenol
2
Western Blot Protein Analysis Protocol
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Protein was extracted from cells using a protein lysis buffer (Beyotime Institute of Biotechnology) immediately after the end of treatment. Total cell protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Equal amounts of protein (10 µg) from the cell lysates were run with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Inc.). Following electrophoresis, proteins were transferred to polyvinylidene membranes (EMD Millipore, Billerica, MA, USA), blocked with 5% fat-free milk at room temperature for 1 h, and incubated with the indicated primary antibodies overnight at 4°C. Subsequently, the membranes were washed with Tris-buffered 0.2% saline-Tween 20 and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:10,000; cat. no. ZDR-5306; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 1 h at room temperature. Immune complexes were detected with enhanced chemiluminescence reagents (EMD Millipore, Boston, MA, USA), and the blots were quantified by densitometric analysis using the Alpha Imager 2200 (Genetic Technologies, Inc., Miami, FL, USA).
3
Microglial Protein Expression Analysis
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Concentrated microglial conditioned
media and cell samples were briefly sonicated and loaded into 12%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad).
After electrophoresis and transfer onto nitrocellulose membranes (GE
Healthcare, Milano, Italy), membranes were blocked for 1 h in blocking
solution PBS—0.1% Tween-20 (Sigma-Aldrich) and 4% nonfat dry
milk (Bio-Rad)—and incubated overnight at 4 °C with primary
antibodies in PBS—0.1% Tween-20. Primary antibodies used were
rabbit anti-iNOS, rabbit anti-IL1β, rabbit anti-TREM2, rabbit
anti-TGFβ2, and mouse anti-GAPDH (all 1:1000 dilution except
for anti-GAPDH used 1:20,000 dilution, all obtained from Santa Cruz
Biotechnology). Membranes were then incubated with specific secondary
antibodies conjugated to horseradish peroxidase (goat anti-rabbit
and goat anti-mouse, both at 1:2000 dilution and from Santa Cruz)
for 90 min at room temperature in PBS—0.1% Tween-20. Labeled
proteins were visualized by using the Clarity Western ECL substrate
(Bio-Rad) and detected using Bio-Rad Image Lab software with a ChemiDoc
MP imaging system (Bio-Rad).
media and cell samples were briefly sonicated and loaded into 12%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad).
After electrophoresis and transfer onto nitrocellulose membranes (GE
Healthcare, Milano, Italy), membranes were blocked for 1 h in blocking
solution PBS—0.1% Tween-20 (Sigma-Aldrich) and 4% nonfat dry
milk (Bio-Rad)—and incubated overnight at 4 °C with primary
antibodies in PBS—0.1% Tween-20. Primary antibodies used were
rabbit anti-iNOS, rabbit anti-IL1β, rabbit anti-TREM2, rabbit
anti-TGFβ2, and mouse anti-GAPDH (all 1:1000 dilution except
for anti-GAPDH used 1:20,000 dilution, all obtained from Santa Cruz
Biotechnology). Membranes were then incubated with specific secondary
antibodies conjugated to horseradish peroxidase (goat anti-rabbit
and goat anti-mouse, both at 1:2000 dilution and from Santa Cruz)
for 90 min at room temperature in PBS—0.1% Tween-20. Labeled
proteins were visualized by using the Clarity Western ECL substrate
(Bio-Rad) and detected using Bio-Rad Image Lab software with a ChemiDoc
MP imaging system (Bio-Rad).
4
Western Blot Analysis of Insulin-Resistant HepG2 Cells
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Standard protocol was followed to prepare lysates of insulin‐resistant HepG2 cells using sample buffer and PMSF. Fifty micrograms of protein, once quantified by modified Bradford protein assay kit, was separated using a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio‐Rad, Hercules, CA). The polyvinylidene difluoride (PVDF) membranes were incubated overnight on a shaker at 4°C with primary antibody prepared in 5% skim milk and visualized on X‐ray film after incubating PVDF membranes with secondary antibody for 2 hr at room temperature. Band intensities were quantitated using CS analyzer software (Atto Corp., Tokyo, Japan).
5
Isolation and Characterization of Synaptic Markers
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Synaptophysin, a presynaptic marker, and PSD95, a postsynaptic marker, were detected in a crude synaptosome fraction by immunoblotting with the specific antibodies. Using an established protocol,34 (link) crude synaptosomes were isolated from neurons (D30 after plating of MGE) generated from UB068 and UB068_CHRFAM7A lines. Briefly, the cells were collected with accutase, and pelleted by centrifugation (300 × g. 5 min, room temperature). The pellet was resuspended in TE buffer (10 mM TRIS, 1 mM EDTA, pH, 7.5) with protease inhibitors, sonicated, and centrifuged (800 × g, 5 min, 4 °C). After centrifugation, total lysate, the supernatant, was further centrifuged (9200 × g, 20 min, 4 °C). The pellet, a crude synaptosome fraction, was resuspended in an appropriate volume of TE buffer with protease inhibitors. 10 μg of crude synaptosomes were separated on 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad), transferred onto polyvinylidene difluoride membrane (Bio-Rad), and incubated overnight at 4 °C with primary antibodies (listed in Supplementary Table S2 ). Specific immunoreactive bands were detected using ChemiDoc XRS + Imaging Systems (Bio-Rad). Densitometry analysis was performed using Image Lab 6.0.1 Software. β-actin was used as a loading control.
6
Western Blot Protein Analysis
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Cells were lysed using radioimmunoprecipitation assay buffer with protease inhibitors. Protein concentration was determined using a DC protein assay kit (Bio-Rad) according to the manufacturer’s protocols. Samples (50 μg) were mixed with the proper amount of 4X sample buffer, analyzed by 4%–20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad), and transferred onto polyvinylidene fluoride membranes. Membranes were incubated with primary and secondary antibodies in PBS containing 0.05% Tween-20 and were then washed three times. After the washes, the immunoblots were developed using the Odyssey Infrared Imaging System (LI-COR Biosciences). The band intensity was analyzed using ImageJ software.
7
Cell Lysis and Protein Analysis
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Total cell lysates from MGE progenitors were prepared using RIPA buffer (Cell Signaling Technologies) according to the manufacturer’s protocol. Twenty-five μg of total protein was separated on 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad), transferred onto polyvinylidene difluoride membrane (Bio-Rad), and incubated overnight at 4 °C with primary antibodies (Supplementary data , Table 2 ). Specific immunoreactive bands were detected using ChemiDoc XRS + Imaging Systems (Bio-Rad).
8
Western Blot Analysis of Protein Targets
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Western blot samples were prepared from the cultured cells that were lysed in RIPA buffer (40 mM Tris-HCl, pH7.5, 1% NP-40, 150 mM NaCl, 2 mM EDTA, 2 mM Na3VO4, 50 mM NaF) containing protease inhibitor cocktail (Roche, Basel, Swiss). The samples were boiled for 5 min at 95°C. Lysates were centrifuged at 13,000 g for 20 min at 4℃. Proteins in the samples were separated in 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA), and transferred to polyvinylidene di uoride membranes (Merck Millipore, Burlington, MA, USA). The proteins were blotted with primary and secondary antibodies. The antibodies used were as follows: NEDD4, HER3, PTEN, phospho-HER3 (pHER3[Y1289]), RAS, ERK1/2, phospho-ERK1/2 (pERK1/2[T202/Y204]), AKT, phospho-AKT (pAKT[S473]) (Cell Signaling Technology, Danvers, MA, USA), ER (Thermo Fisher Scienti c) and β actin (Sigma-Aldrich). Expression levels of the proteins were visualized by SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scienti c) and ChemiDoc XRS Plus (Bio-Rad).
9
Western Blot Analysis of Protein Expression
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Total protein from cells was extracted using RIPA lysis buffer containing protease inhibitor. The protein content in supernatant was detected via bicinchoninic acid method. Next, an equal volume of protein (50 mg) was loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA) and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Then the membranes were incubated in tris-buffered saline tween with 5% skim milk at room temperature to block non-specific binding. After that, the membranes were incubated with primary antibodies at 4 °C overnight, and further with secondary antibody at room temperature for 1 h. Then the protein bands were visualized and imaged using the BioSpectrum system (Bio-Rad). The antibodies are shown in Table 3 .
Antibodies for western blot assay
| Antibody | Item No. | Dilution ratio |
|---|---|---|
| E-cadherin | ab1416 | 1: 100 |
| Vimentin | ab119139 | 1: 100 |
| MDM2 | ab38618 | 1: 5000 |
| Snail | ab53519 | 1: 50 |
| β-actin | ab179467 | 1: 5000 |
| Smad 2 | ab33875 | 1:2000 |
| Smad 3 | ab40854 | 1:2000 |
| Secondary antibody | ab150117 | 1: 5000 |
Note: all antibodies were purchased from Abcam Inc., Cambridge, MA, USA
10
TGFBI Protein Expression Analysis
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RIPA lysis buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), which contained a protease inhibitor, was used to lyse protein from the cells. We separated proteins by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The TGFBI expression levels were determined by western blotting. We normalized the protein levels by probing the same blots with a GAPDH antibody and analyzed the protein bands using Bandscan software Image J. The TGFBI antibody (A2561) was purchased from ABclonal Biotechnology (Woburn, MA, USA), and the GAPDH antibody (FL-335, sc-25778) was purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA).
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