Melanoma A375 cells were harvested and homogenized in lysis buffer containing phenylmethylsulfonyl fluoride (PMSF). The primary antibodies used were p75NTR (#ab52987; Abcam, Cambridge, UK), NF‐κB p65 (#8242; CST, Danvers, MA, USA), IκBα (#9242; CST, Danvers, MA, USA), Phospho‐IκBα (#2859; CST, Danvers, MA, USA), caspase‐3 (ab214430; Abcam, Cambridge, UK), caspase‐9 (ab202068; Abcam, Cambridge, UK), Bax (ab32503; Abcam, Cambridge, UK), Bcl‐2 (ab32124; Abcam, Cambridge, UK), Lamin B1 (Beyotime, Shanghai, China), inhibitor of apoptosis protein 2 (c‐IAP2; #380798; ZEN BIO, Chengdu, China), bFGF (#381676; ZEN BIO, Chengdu, China) and β‐actin (Sino Biological, Beijing, China). β‐actin or Lamin B1 expression served as an internal control. For immunofluorescence analysis, cells were incubated with anti‐(human NF‐κB p65) IgG (1 : 100). The primary antibody was detected using a Cy3‐labeled IgG (1 : 100; Beyotime, Shanghai, China), and nuclei were stained with 4',6‐diamidino‐2‐phenylindole dihydrochloride (DAPI). Microscopic analysis was performed using a Nikon E600 microscope (Tokyo, Japan).
β actin
Manufactured by Sino Biological
Sourced in China, United States
β-actin is a cytoskeletal protein that plays a crucial role in cell structure and function. It is a highly conserved protein found in all eukaryotic cells and is often used as a reference gene or loading control in various molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Sino Biological (2 mentions)
Bca protein quantification kit, by Vazyme (2 mentions)
Ab214430, by Abcam (1 mentions)
Ab52987, by Abcam (1 mentions)
Ab202068, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Lamin b1, by Beyotime (1 mentions)
E600 microscope, by Nikon (1 mentions)
Ab32124, by Abcam (1 mentions)
Proteinase inhibitor cocktail, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Flag tag (flag), by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Hrp conjugated affinipure goat anti mouse igg h l, by Proteintech (1 mentions)
Cell lysis reagent, by Merck Group (1 mentions)
Mitochondria cytosol fractionation kit, by Abcam (1 mentions)
Rassf1a, by Abcam (1 mentions)
13 protocols using β actin
1
Melanoma A375 Cell Signaling Pathway
2
Protein Expression Analysis in Ischemic Brain
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Total proteins from ischemic brain tissue and primary neurons were extracted by tissue homogenization in RIPA buffer containing a proteinase inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, USA). Equal amounts of lysed and boiled protein (30 μg/well) were separated on 12% SDS-PAGE gel and proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. Blots were probed with anti-Bax (Abcam, ab32503), anti-p53 (MultiSciences Biotech, Hangzhou, China, ab37355–100), anti-PUMA (MultiSciences Biotech, China, ab40081–100), anti-AKT (Cell Signaling Technology, 2920s), and anti-phospho-AKT (Cell Signaling Technology, 4060), and then detected using HRP-conjugated secondary antibody (Abcam). β-actin (Sino Biological Inc, Beijing, China, 100166-MM10) was used as a loading control. The protein bands were visualized with enhanced chemiluminescence reagents, and the resulting images were analyzed using ImageJ and normalized to β-actin.
3
Western Blot Analysis of LSD1 and FLAG
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The cells were collected and lysed at 4 °C with RIPA (Solarbio, R0010) buffer containing protease and phosphatase inhibitors. Protein lysates were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to PVDF membranes (Millipore, ISEQ00010). Membranes were incubated in blocking buffer (5% milk, 0.1% Tween20 in Tris-buffered saline) for 1 h and probed overnight with primary antibodies at 4 °C, followed by incubation with the corresponding HRP-conjugated secondary antibodies. Pierce ECL Plus Western blotting substrate kit (Tanon, 180–5001). The antibodies were used according to the manufacturer’s recommendations were used. Antibodies used for Western blot analyses were as follows: LSD1 (Cell Signaling Technology, 2139S), FLAG (Cell Signaling Technology, #14793), β-actin (Sino Biological, 1,000,166-MM10), Goat anti-rabbit IgG H&L (HRP) (Abcam, ab6721), HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (ProteinTech Group, Inc., SA00001-1).
4
Cytosolic Protein Analysis by Western Blot
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Total cellular protein samples were prepared with a cell lysis reagent (Sigma–Aldrich) according to the manual and supplemented with a protease inhibitor cocktail (Pierce, Rockford, IL, USA). For the Cyt C release assay, the cytoplasmic protein was isolated by the Mitochondria/Cytosol Fractionation Kit (Abcam, Cambridge, UK). Each protein sample was separated by 10% SDS-PAGE gel and was transferred to a nitrocellulose membrane. Then the rabbit polyclonal antibody to RARβ2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), RASSF1A (Abcam, Cambridge, UK), caspase 3 (Sino Biological, Beijing, China), cleaved-PARP (Cell Signaling Technology Inc., Danvers, MA, USA), Cytochrome c (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or β-actin (Sino Biological, Beijing, China) was used to detect the protein level of each molecule. Goat anti-rabbit IgG conjugated to horseradish peroxidase (Pierce) and ECL detection systems (Super Signal West Femto; Pierce) were used for detection.
5
Influenza Virus Infection Assay in MDCK Cells
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One day after
seeding of MDCK
cells in six-well plates (6 × 105 cells per well),
they were mock-infected or infected with PR8 virus at an MOI of 0.01
for 1 h at 35 °C. After removal of unadsorbed influenza virus,
increasing concentrations of each compound were used to treat cells
for an additional day at the same temperature. Cell lysates harvested
using M-PER reagent (Thermo Fisher Scientific, Rockford, IL, USA)
were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), followed by electrotransfer to polyvinylidene fluoride
membranes (Merck-Millipore, Tullagreen, Ireland). Membranes were probed
with antibodies raised against viral NP (Cat. no., 11675-T62; Sino
Biological, Beijing, China) and β-actin as a loading control
(Cat. no., A1798; Sigma-Aldrich) according to previously described
protocols.34 (link)
seeding of MDCK
cells in six-well plates (6 × 105 cells per well),
they were mock-infected or infected with PR8 virus at an MOI of 0.01
for 1 h at 35 °C. After removal of unadsorbed influenza virus,
increasing concentrations of each compound were used to treat cells
for an additional day at the same temperature. Cell lysates harvested
using M-PER reagent (Thermo Fisher Scientific, Rockford, IL, USA)
were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), followed by electrotransfer to polyvinylidene fluoride
membranes (Merck-Millipore, Tullagreen, Ireland). Membranes were probed
with antibodies raised against viral NP (Cat. no., 11675-T62; Sino
Biological, Beijing, China) and β-actin as a loading control
(Cat. no., A1798; Sigma-Aldrich) according to previously described
protocols.34 (link)
6
Protein Expression Analysis in Rat Liver
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The concentration of protein extracted from rat liver using RIPA buffer was then determined using the BCA assay. SDSPAGE was employed to separate the proteins, followed by transfer onto a PVDF membranes. The membrane was then incubated with a blocking buffer containing 5% skimmed milk for 1 h, washed with TBST three times, and subsequently incubated overnight with primary antibodies including βactin (Sino Biological), Desmin (Boster), Tagln (Boster), Tpm2 (Sangon Biotech), Csrp1 (Sangon Biotech), and Myh11(Cusabio) at dilutions of 1:1000, 1:400, 1:500, 1:300, 1:500, and 1:300, respectively, at 4 °C. After washing three times with TBST, the membrane was incubated with secondary antibodies for 1 h, followed by staining. Results were visualized using a Chemiscope 3000 (Clinx Science Instruments Co., Ltd).
7
Western Blot for Protein Detection
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Protein was extracted from cells and transferred to polyvinylidene fluoride (PVDF) membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, membranes were blocked with 5% nonfat milk for 1 hour and incubated overnight at 4° C with the following primary antibodies: YWHAZ (1 : 500, Abcam, ab51129) and β-actin (38 KD, Sino Biological Inc, 100166-MM1). After washing the membranes three times with Tris Buffered Solution Tween (TBST), a secondary antibody was incubated with the membranes for 1 hour at room temperature. The dilution ratio was determined according to the instructions.
8
Dietary ALA Modulates Metabolic Factors
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α-Linolenic acid (ALA) (97%) was obtained from Shanghai Guchen Biological (Shanghai, China). A normal chow diet (NCD) (catalog number AIN-93M) and a 60% high-fat diet (catalog number TP23400) were purchased from Trophic Animal Feed High-tech Co., Ltd., Nantong, China. Triglycerides (TG), serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and liver TG were measured using kits from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Plasma insulin was assessed using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) kit (Alpco, USA). Serum lipopolysaccharide (LPS) levels were quantified using an ELISA kit (Cusabio, USA). Serum TNF-α, IL-6, and IL-1β were measured using enzymatic kits purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China). Antibodies were purchased from Abcam (occludin, catalog number ab167161), Thermo Fisher (ZO-1, catalog number 61-7300), Wanleibio (TLR4, catalog number WL00196), and Sino Biological (β-actin, catalog number SB100166-MM10).
9
Western Blot Analysis of Protein Expression
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CNE-2 cells were lysed with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) and supplemented with protease inhibitor cocktail kit (Roche Biochemicals, Penzberg, Germany). Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. The membrane was incubated with primary antibodies for LMP1- (1:2000, Abcam, Cambridge, UK), LMP2A- (1: 1000, Abcam), eIF4E-specific (1: 1000, Abcam) or β-actin (1: 3000, Sino Biological, Wayne, PA, USA). Horseradish peroxidase (HRP)-linked secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) and enhanced chemiluminescence (Thermo Fisher Scientific) were utilized to visualize the specific binding of the HRP-linked second antibody to first antibody-protein complexe, under the UVP BioSpectrum 500 imaging system (UVP, Upland, CA, USA).
10
Western Blot Analysis of Protein Signaling
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Cells were washed with cold PBS, lysed with RIPA buffer, and kept on ice for 1 h. The protein was incubated by centrifugation at 12,000 rpm, 4 °C for 10 min, and its concentration was quantified using a Bicinchoninic Acid assay with an Easy II Protein Quantitative kit (Beijing TransGen Biotech Co., Ltd., Beijing, China). Protein samples were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% nonfat milk (prepared with TBS-T) for 1 h at room temperature. Membranes were incubated overnight at 4 °C primary antibody (β-actin (Sino Biological, China; 1:1000), PKA (Abcam, USA; 1:1000), CREB (Abcam, USA; 1:1000), p-CREB (Abcam, USA; 1:800), Trkb (Abcam, USA; 1:1000), and p-Trkb (Abcam, USA; 1:800)). The membranes were washed and incubated with a secondary antibody at room temperature for 1 h. Protein expression was visualized using chemiluminescence on the Bio-Rad system.
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