The concentration of total protein extracted from MCF-10A and MDA-MB-231 cells was determined using a BCA Protein Assay Kit (Pierce, USA). Equal amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes (Pierce) using a mini trans-blot (Bio-Rad Laboratories, Hercules, CA, USA). Goat anti-human C35 (MIEN1), AKT, MMP-9, and b-actin were purchased from Santa Cruz Biotechnology, Inc. The b-actin was used as an internal control. Electrochemiluminescence was carried out according to the manufacturer instructions and read with a ChemiImager 5500 imaging system (Alpha Innotech Co., San Leandro, CA, USA).
β actin
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom, China, Germany, Canada, Morocco, Japan, Italy, Switzerland, France, Israel, Singapore, Hong Kong, Sweden, Macao, Panama
β-actin is a cytoskeletal protein that is ubiquitously expressed in eukaryotic cells. It is a component of the microfilament system and plays a crucial role in various cellular processes, such as cell motility, maintenance of cell shape, and intracellular trafficking.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (262 mentions)
Bcl 2, by Santa Cruz Biotechnology (239 mentions)
Gapdh, by Santa Cruz Biotechnology (229 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (197 mentions)
Cleaved caspase 3, by Cell Signaling Technology (174 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (164 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (139 mentions)
P akt, by Cell Signaling Technology (138 mentions)
Cyclin d1, by Santa Cruz Biotechnology (128 mentions)
Caspase 3, by Cell Signaling Technology (113 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (112 mentions)
Caspase 3, by Santa Cruz Biotechnology (105 mentions)
P erk, by Cell Signaling Technology (104 mentions)
Protease inhibitor cocktail, by Roche (93 mentions)
E cadherin, by Santa Cruz Biotechnology (92 mentions)
P akt, by Santa Cruz Biotechnology (88 mentions)
P erk, by Santa Cruz Biotechnology (87 mentions)
Polyvinylidene difluoride membrane, by Merck Group (87 mentions)
Bcl 2, by Cell Signaling Technology (87 mentions)
Ripa buffer, by Thermo Fisher Scientific (87 mentions)
5 071 protocols using β actin
1
Protein Expression Analysis of Breast Cell Lines
2
Analyzing β-Catenin Signaling in hADMSCs
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A western blotting assay was applied to study the protein expressions of hADMSCs related to the β-catenin signaling pathway. Proteins were extracted and subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) for the detection of active-form β-catenin, then transferred to PVDF membranes. The membranes were blocked with 5% skim milk in tris-buffered saline with Tween 20 for 1 hour at room temperature. Membranes were reacted with primary antibodies overnight at 4°C. Primary antibodies were as follows: anti-nonphosphorylated β-catenin (1:500 [EMD Millipore, Billerica, MA, USA]) and β-actin (1:1000 [Santa Cruz Biotechnology Inc., Dallas, TX, USA]), and the β-actin protein was used as the control.
3
Western Blot Analysis of MHC and Oncogene Expression
4
Western Blot Analysis of Brain Proteins
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The brain cortices were homogenized in ice-cold lysis buffer (Beyotime) with PMSF (final concentration 1 mM) for 15 min and centrifuged at 12,000 rpm at 4°C for 10 min. The supernatants were collected and boiled with 5x sample buffer at 95°C for 5 min. Then the samples were separated by SDS/PAGE and transferred onto PVDF membrane at 350 mA for 55 min at 4°C. The membranes were blocked with 10% horse serum in 0.01 M PBS for 2 h, probed with AQP4 (1 : 200, rabbit polyclonal antibody, Chemicon), α-DG and β-DG (1 : 200), and β-actin (1 : 5000, mouse monoclonal antibody, Santa Cruz) antibody overnight at 4°C. The blots were incubated with horse radish peroxidase conjugated antibody (1 : 10000 for β-actin, 1 : 500 for other antibodies, Santa Cruz) for 4 h. The specific reaction was visualized by using a chemiluminescent substrate (Pierce, USA). The bands were quantified by gel densitometry (Bio-Rad, Hercules, USA). The value of individual protein band was divided by the value of β-actin from the same sample; a ratio of protein/β-actin for each sample was obtained.
5
Histone Modification Analysis by Western Blot
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Whole-cell lysate was prepared in 3D-RIPA buffer. Protein (20 µg) was separated by 12% SDS–PAGE and transferred to a polyvinylidene difluoride membrane. Membranes were blocked using 5% nonfat dry milk in PBS containing 0.05% Tween 20, and then incubated with primary antibody overnight at 4 °C. HRP-conjugated secondary antibody was used and detected using the Pierce ECL Western Blot Substrate. Antibodies were obtained from the following sources: H3K9Ac (Abcam, ab32129, 1:1000), H3K4Me2 (Abcam, ab32356, 1:5000), H3K4Me1 (Abcam, ab8895, 1:5000), H3K4Me3 (Abcam, ab8580, 1:5000), H3K14Ac (Millipore, 07–353, 1:5000), H3K18Ac (Millipore, 07–354, 1:5000), LSD1 (Abcam, ab17721, 1:500), HDAC1 (Abcam, ab19845, 1:1000), total H3 (Cell Signaling Technology, 4499 S, 1:1000 or Abcam, ab1791, 1:10,000), β-actin (Santa Cruz Biotechnology, sc47778, 1:2000), CoREST1 (BD Transduction Laboratories, 612146, 1:500), SIN3A (Abcam, ab129087, 1:1000), HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, 1:5000 for β-actin blots, 1:2000 for all others). Blots shown are representative of at least two independent experiments. Uncropped versions of Western blots are provided in the Supporting Information (Supplementary Figs. 32 and 33 ).
6
Western Blot Analysis of CDKN2A/p16INK4a
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Total cell lysates were harvested in RIPA buffer (150mM NaCl, 1% Nonidet P-40, 50mM
Tris, pH8.0, protease inhibitor cocktail) and protein concentrations were determined by Bradford protein assay (Bio-Rad Laboratories, Inc. Hercules, CA, USA) and absorbance was measured at 595 nm on the Tecan Infinite® 2000 PRO plate reader (Tecan, Seestrasse, Switzerland). Equal amounts of proteins from each lysate were mixed with 3X loading dye and heated at 95 °C for 10 minutes. The samples were resolved by 12% SDS-PAGE (running buffer: 25 mM Tris, 192 mM Glycine, and 0.1% SDS) and then transferred to PVDF membranes (transfer buffer: 25 mM Tris, 192 mM Glycine, and 20% (v/v) methanol (Fischer Chemical)). Membranes were blocked with TBST buffer containing 5% skim milk one hour at room temperature with gentle shaking. The blocked membranes were further washed three times with TBST buffer and incubated at 4°C overnight with primary antibodies CDKN2A/p16INK4a (ab108349, Abcam, 1:1000), β-actin (Santa Cruz β-actin (C4) Mouse monoclonal IgG1 #sc-47778, 1:5000), followed by HRPconjugated secondary antibody incubation at room temperature for one hour. Both the primary and secondary antibodies were diluted in 5% BSA-TBST buffer, and all the incubations were performed in a gentle shaking manner. The immune-reactive proteins were detected using the Luminata Crescendo Western HRP substrate (Millipore).
Tris, pH8.0, protease inhibitor cocktail) and protein concentrations were determined by Bradford protein assay (Bio-Rad Laboratories, Inc. Hercules, CA, USA) and absorbance was measured at 595 nm on the Tecan Infinite® 2000 PRO plate reader (Tecan, Seestrasse, Switzerland). Equal amounts of proteins from each lysate were mixed with 3X loading dye and heated at 95 °C for 10 minutes. The samples were resolved by 12% SDS-PAGE (running buffer: 25 mM Tris, 192 mM Glycine, and 0.1% SDS) and then transferred to PVDF membranes (transfer buffer: 25 mM Tris, 192 mM Glycine, and 20% (v/v) methanol (Fischer Chemical)). Membranes were blocked with TBST buffer containing 5% skim milk one hour at room temperature with gentle shaking. The blocked membranes were further washed three times with TBST buffer and incubated at 4°C overnight with primary antibodies CDKN2A/p16INK4a (ab108349, Abcam, 1:1000), β-actin (Santa Cruz β-actin (C4) Mouse monoclonal IgG1 #sc-47778, 1:5000), followed by HRPconjugated secondary antibody incubation at room temperature for one hour. Both the primary and secondary antibodies were diluted in 5% BSA-TBST buffer, and all the incubations were performed in a gentle shaking manner. The immune-reactive proteins were detected using the Luminata Crescendo Western HRP substrate (Millipore).
7
Western Blotting and Cellular Fractionation
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Western blotting was performed according to a method described previously [18 (link)]. The preparation of cytosolic and nuclear fractions of cells was performed according to the procedures described by Vasanthi et al [22 (link)]. Protein extracts were subjected to centrifugation at 10,000g for 10 minutes. Total protein (10–50 μg per lane) was electrophoresed and separated on 8–15% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After blocking with 5% nonfat dry milk, the membranes were incubated with the indicated primary antibodies overnight at 4°C. The blots were incubated with the antibodies against NF-κB, C23, IκBα, caspase-3, caspase-8, caspase-9, poly (ADP-ribose) polymerase-1 (PARP-1), and β-actin, purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); β-actin served as an internal control. The blot was quantified by enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK).
8
Western Blot Analysis of Lung Proteins
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Total protein from right upper lung tissues was extracted by using a commercially available kit (KGP250; Nanjing Keygen Biotech Co. Ltd., China). Protein concentrations were determined by the BCA method. Equal amount of protein extract was separated by electrophoresis on 10% polyacrylamide SDS gels and transferred to a PVDF membrane. The membranes were blocked with 5% nonfat milk for 60 min and then probed with antibodies to IL-1β, TNF-α, β-actin (1:1,000; all from Santa Cruz Biotechnology), EGFR, AKT, ERK1/2, TLR4, p65 (1:1,000; all from Abcam, UK), p-EGFR-Tyr1068, p-AKT-Thr308, p-ERK-Thr202/Tyr204, p-p65-Ser536 (1:1,000; all from Cell Signaling Technology). After overnight incubation with the primary antibodies, the membranes were incubated with anti-rabbit IgG HRP-conjugated or anti-mouse lgG HRP-conjugated (1:3,000; all from Santa Cruz Biotechnology) for 1 h. Normalization for protein determination was carried out with βactin. The immunoreactive bands were visualized and photographed by using an EC3 Imaging System (UVP Inc.).
9
Western Blot Analysis of Autophagy Markers
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Antibodies were obtained from commercial sources: anti-NMDAR1, anti-NMDAR2B, and anti-LC3A/B; anti-APG5 and anti-ULK1 (Abcam, Cambridge, UK); anti-beclin1, anti-AMP-activated protein kinase (AMPK) α, and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA).
For the western blot analysis, the cells were rinsed with phosphate-buffered saline (PBS) and subsequently lysed for 30 min on ice in RIPA-B buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 50 mM NaF, 100 μM Na3VO4, 1 mM DTT, and 50 μg/mL PMSF). The insoluble material was removed by centrifugation at 12000 rpm for 20 min. Next, the supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blots were blocked in PBS with 5% skim milk and 0.05% Tween 20, incubated with the diluted primary antibodies against NMDAR1, NMDAR2B, or LC3A/B (1: 1000; Cell Signaling Technology, Denvers, MA, USA); APG5 or ULK1 (1: 1000; Abcam, Cambridge, UK); beclin-1 or AMPKα (1: 200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and followed by incubation with a horseradish peroxidase-conjugated secondary antibodies (1: 2500; Santa Cruz). β-actin (Santa Cruz) was used as a control. The blots were assayed using an enhanced chemiluminescence detection system (Image Quant LAS 4000mini, Pittsburgh, PA, USA).
For the western blot analysis, the cells were rinsed with phosphate-buffered saline (PBS) and subsequently lysed for 30 min on ice in RIPA-B buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 50 mM NaF, 100 μM Na3VO4, 1 mM DTT, and 50 μg/mL PMSF). The insoluble material was removed by centrifugation at 12000 rpm for 20 min. Next, the supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blots were blocked in PBS with 5% skim milk and 0.05% Tween 20, incubated with the diluted primary antibodies against NMDAR1, NMDAR2B, or LC3A/B (1: 1000; Cell Signaling Technology, Denvers, MA, USA); APG5 or ULK1 (1: 1000; Abcam, Cambridge, UK); beclin-1 or AMPKα (1: 200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and followed by incubation with a horseradish peroxidase-conjugated secondary antibodies (1: 2500; Santa Cruz). β-actin (Santa Cruz) was used as a control. The blots were assayed using an enhanced chemiluminescence detection system (Image Quant LAS 4000mini, Pittsburgh, PA, USA).
10
Western Blot Analysis of Cellular Proteins
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Total proteins were extracted using RIPA buffer and Western-blot performed as previously described [22] (link). The following primary antibodies directed against p21 (sc-397), β-actin (sc-47,778), γH2AX (sc-101,696), RIP1 (sc-7881) and HIF-2 (sc28706) from Santa Cruz Biotechnology and that against PARP (#5246) and beclin-1 (#3495) from Cell signaling. Antibody against HIF-1 (#610,959) was purchased from BD Sciences. Beta Actin (sc-47,778, Santa Cruz) was used to verify that similar protein amounts were loaded in all lanes.
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