PHGDH (HPA021241, 1:2000 for western blot, 1:4000 or 1:8000 for IHC for the Norwegian and Dutch cohort, respectively) and PSPH (HPA020376, 1:1000 for western blot) antibodies were purchased from Sigma-Aldrich. The PSAT1 (CPTC-PSAT1-2, 1:500 for western blot) and PARP1 (AFFN-PARP1-17B10, 1:150 for western blot) antibodies, developed by the National Cancer Institute and EMBL MACF, respectively, were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. The phospho-Histone H2A.X (Ser139) (20E3, 1: 600 for IF), ATF4 (D4B8, 1:1000 for WESTERN BLOT) and β-actin (13E5, 1:5000, for western blot) antibodies were purchased from Cell Signaling Technology. Anti-p53 Antibody [DO-1]-Chip graded was obtained from Abcam (ab1101, 1:200 for IHC) while PAX8 Polyclonal antibody was obtained from Proteintech (10336-1-AP, 1:1200 for IHC). Secondary peroxidase conjugated goat anti-rabbit (111-035-003, 1:5000 for western blot (1:10 000 for β-actin western blot)) and goat anti-mouse antibodies (115-035-044, 1:10,000 for western blot) were purchased from Jackson ImmunoResearch. Secondary donkey anti-rabbit Alexa Fluor 594 (1:800) was purchased from Molecular Probes, Life Technologies.
β actin
Manufactured by Jackson ImmunoResearch
Sourced in United Kingdom, United States
β-actin is a cytoskeletal protein that is widely used as a loading control or reference gene in various biochemical and cell-based assays. It is a highly conserved and abundantly expressed protein that is involved in the structural integrity and motility of cells.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Hpa021241, by Merck Group (1 mentions)
Phospho histone h2a x ser139 20e3, by Cell Signaling Technology (1 mentions)
Phgdh, by Merck Group (1 mentions)
Hpa020376, by Merck Group (1 mentions)
Atf 4 d4b8, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Integrin β1, by Merck Group (1 mentions)
Gapdh sc 47724, by Santa Cruz Biotechnology (1 mentions)
N cadherin ab19348, by Abcam (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Cyclin b1, by Merck Group (1 mentions)
Cyclin d1 ab16663, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
E cadherin, by BD (1 mentions)
Dylight 488 donkey anti mouse, by Jackson ImmunoResearch (1 mentions)
P egfr, by Cell Signaling Technology (1 mentions)
Image studio, by LI COR (1 mentions)
Hybond nitrocellulose membrane, by Cytiva (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
5 protocols using β actin
1
Antibody Validation for Western Blot and IHC
2
Comprehensive Protein Expression Analysis
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Western blotting and indirect immunofluorescence were performed using the following primary antibodies: β actin (A5441, Sigma-Aldrich, Ireland); β1 integrin (#1952, Merck Millipore, Ireland); cyclin B1 (#4135), estrogen receptor α (#8644), HER2 (#4290), EGFR (#4267), IGF-1R (#3027), pan AKT (#2920), pan ERK (#4696), pan p-AKT (#4060), pan p-ERK (#4377), p-EGFR (#3777), progesterone receptor A/B (#8757) [all from Cell Signaling Technology Europe, Leiden, Netherlands]; cyclin D1 (ab16663) and N-cadherin (ab19348) [both from Abcam, Cambridge, UK]; E-cadherin (#610181, BD Biosciences, San Jose, CA, USA); GAPDH (sc47724) and vimentin (sc32322) [both from Santa Cruz Biotechnology, Dallas, TX, USA]. Secondary antibodies for western blots were IRDye 800CW or 680RD (LI-COR Biosciences UK Ltd., Cambridge, UK); and the secondary antibody used for β actin indirect immunofluorescence was DyLight 488 donkey anti-mouse (Jackson ImmunoResearch Europe Ltd., Ely, UK).
3
Western Blot Analysis of Cell Signaling
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Cells (1 × 106/condition) were harvested by centrifuging at 2,500 rpm for 5 min at 4°C and washing with 1 mL of ice-cold PBS. Cell pellets were resuspended in 50 μL of ice-cold PBS and 50 μL of 2x SDS Sample Buffer (50 mM Tris, 2% β-mercaptoethanol, 2% SDS, 0.04% bromophenol blue, 10% glycerol, pH 6.8) and boiled for 10 min at 95°C. Cells (200,000 per condition) were analyzed on 8% resolving gels by SDS-PAGE, and proteins were transferred to PVDF membrane. The membranes were blocked with 5% milk in TBS-T and incubated with primary antibodies overnight with agitation in the cold room. GATA2 antibody was described previously.64 (link) The following antibodies were from Cell Signaling: p-STAT1 (9167), STAT1 (14,995), p-STAT3 (9145), STAT3 (12,640), p-ERK1/2 (9101), ERK1/2 (9102), p-p38 (9216), p38 (8690), p-JNK (9255), JNK (9252), α-tubulin (3873), and β-actin (3700), and secondary antibodies were from Jackson Laboratory. Proteins were detected and quantified with chemoluminescence (FEMTO supersignal, Pierce) using LI-COR imaging instrument, and densitometry analysis was performed with Image Studio.
4
Western Blot Analysis of Lactoferrin
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Proteins were denatured in sample buffer and separated on a 10% SDS-PAGE and electroblotted into Hybond nitrocellulose membranes (Amersham, Baie D’Urfé, Canada). The following antibodies were used for immunoblotting: 1:2000 anti-human Lf (78 kDa; Santa Cruz Biotechnology, USA), 1:40000 anti-β-actin (42 kDa; Sigma, USA) and 1/10000 horseradish peroxidase-conjugated secondary antibodies (Jackson Laboratory, USA), The β-actin protein expression was determined to confirm equal loading.
5
Western Blot Analysis of Liver Tissue
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Briefly, liver tissue was harvested and extracted by RIPA lysis buffer (Thermo Fisher). After protein BCA quantification (Thermo Fisher), the samples were separated by SDS-PAGE gel electrophoresis and immunoblotted to nitrocellulose membranes. Subsequently, the membranes obtained were blocked with 5% milk and incubated with 5% BSA dissolved antibodies overnight at 4°C. The antibodies used in this study are detailed as follows: β-actin, anti-Rabbit (all from Jackson, 1 : 10000), phosphor-p38 (CST,4511), p-38 (CST, 8690), phosphor-JNK1(CST, 9259), JNK1(CST, 3708), and RIP1(CST, 3708) (all from Cell Signaling Technology, 1 : 1000). Besides, the second antibodies were diluted in 5% BSA and incubated for 1 hour at room temperature.
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