Protein was extracted from liver tissue or cell cultures as described.2 (link) Protein was extracted from liver tissue or cell cultures with ice-cold protein lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, and 1% Triton-100). Proteins (30 µg/sample) in SDS-loading buffer (50 mM Tris, pH 7.6, 10% glycerol, and 1% SDS) were subjected to 4–20% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% dry milk and 0.1% Tween 20 (USB, Cleveland, OH). Monoclonal rabbit anti-mouse NICD, HSF1, Snail, TRX1, TXNIP, NLRP3, ASC, cleaved caspase-1, p-ASK1, ASK1, Bcl-2, Bcl-xL, cleaved caspase-3, and β-actin Abs (Cell Signaling Technology, MA) were used. The membranes were incubated with the Abs and then developed according to the Pierce SuperSignal West Pico Chemiluminescent Substrate protocol (Pierce Biotechnology, Rockford, IL). The relative quantities of protein were determined using a densitometer (Kodak Digital Science 1D Analysis Software, Rochester, NY) and expressed in absorbance units (AU) by comparison with β-actin expression.
β actin ab
Manufactured by Cell Signaling Technology
Sourced in Canada, United States, Japan
β-actin Abs is a primary antibody that binds to the β-actin protein, a ubiquitously expressed cytoskeletal protein. It is commonly used as a loading control in Western blotting and other protein analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Bcl xl, by Cell Signaling Technology (3 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Cleaved caspase 1, by Cell Signaling Technology (2 mentions)
Digital science 1d analysis software, by Kodak (2 mentions)
Pierce supersignal west pico chemiluminescent substrate protocol, by Thermo Fisher Scientific (2 mentions)
Nf κb, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
P ask1, by Cell Signaling Technology (1 mentions)
Phos stat3, by Cell Signaling Technology (1 mentions)
Foxo1, by Cell Signaling Technology (1 mentions)
Phos mtor, by Cell Signaling Technology (1 mentions)
Phos p70s6k, by Cell Signaling Technology (1 mentions)
Hmgb1, by Cell Signaling Technology (1 mentions)
Hif 1α, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Proteinase and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Rock1, by Cell Signaling Technology (1 mentions)
12 protocols using β actin ab
1
Protein Extraction and Quantification from Liver Tissue
2
Western Blot Analysis of Cell Signaling
3
Liver Protein Extraction and Immunoblotting
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Protein was extracted from liver tissue or cell cultures, as described53 (link). Monoclonal rabbit anti-mouse ATF3 (Santa Cruz Biotechnology, Shanghai, China), phos-mTOR, mTOR, phos-p70S6K, p70S6K, HMGB1, TLR4, NF-κB, HIF-1α, PHD1, Bcl-2, Bcl-xl, and β-actin Abs (Cell Signaling Technology, San Diego, CA) were used. The relative quantities of proteins were determined by densitometer, and expressed in absorbance units (AU).
4
Western Blot Analysis of Protein Targets
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Protein was extracted from macrophages or lung tissues with ice-cold protein lysis buffer (50 mM Tris, 150 mM Nacl, 0.1 % sodium dodecyl sulfate, 1 % sodium deoxycholate, 1 % Triton-100). The buffer contains 1 % proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich). Proteins (30 µg/sample) in SDS-loading buffer (50 mM Tris, pH 7.6, 10 % glycerol, 1 % SDS) were subjected to 4–20 % SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5 % dry milk and 0.1 % Tween 20 (USB, Cleveland, OH). Monoclonal rabbit anti-mouse HMGB1, PTEN, phos-β-catenin (Ser552), β-catenin, phos-Akt (Ser473), Foxo1, TLR4, NF-κB and β-actin Abs (Cell Signaling Technology, MA) were used. The membranes were incubated with Abs and then developed according to the Pierce SuperSignal West Pico Chemiluminescent Substrate protocol (Pierce Biotechnology, Rockford, IL). Relative quantities of protein were determined and expressed in absorbance units (AU) comparing to β-actin expression using a densitometer (Kodak Digital Science 1D Analysis Software, Rochester, NY).
5
Protein Extraction and Antibody Analysis
6
Multiprotein Analysis in Liver Samples
7
Protein Extraction and Fractionation from Liver
8
Antibody Procurement for GST Subtypes
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GSTM1 (1H4F2), GSTM2 (E-9), GSTM4 (PL-B12), and GSTK1 (E-4) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). β-actin Ab was purchased from Cell Signaling Technology (Danvers, MA, USA). GSTM3 Ab was procured from ProteinTech Group (Rosemont, IL, USA). GSTP1 and GSTM5 Ab were from Genetex (Irvine, CA, USA).
9
Signaling Pathway Regulation in Cells
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Dimethyl sulfoxide (DMSO), cycloheximide (CHX), N-acetyl-L-cysteine (NAC), fumonisin B1, desipramine and GW4869 were purchased from Sigma-Aldrich (St Louis, MO, USA). The fluorescence-labeled monoclonal antibody (mAb) anti-human TLR2-phycoerythrin (TLR2-PE) and TLR4-PE were purchased from eBioscience (San Diego, CA, USA). The mouse IgG2a-PE was purchased from Becton Dickinson (San Jose, CA, USA). Stress-activated protein kinases (SAPK)/c-Jun N-terminal kinases (JNK) rabbit Ab (#9252), phospho-SAPK/JNK (Thr183/Tyr185) mouse mAb (#9255), p44/42 mitogen–activated protein kinase (MAPK) (extracellular signal-regulated kinase (ERK)1/2) rabbit mAb (#4695), phospho-p44/42 MAPK (Thr202/Tyr204) rabbit mAb (#4370), apoptosis signal–regulating kinase 1 (ASK1) rabbit Ab (#3762), phospho-ASK1 (Thr845) rabbit Ab, phospho-c-Jun (Ser63) rabbit mAb (#2361), phopho-MAPK kinase 7 (MKK7) (Ser271/Thr275) rabbit Ab (#4171), β-actin Ab (#4967), anti-rabbit IgG horseradish peroxidase (HRP)-linked Ab (#7074) and anti-mouse IgG HRP-linked Ab (#7076) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). The fluorescent probe 3′-(p-hydroxyphenyl) fluorescein (HPF) was obtained from Molecular Probes (Eugene, OR, USA).
10
Protein Isolation and Western Blotting
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After cellular proteins isolation using a Protease Inhibitor Cocktail and Protein Phosphatase Inhibitor (Beijing Solarbio Science & Technology Co., Ltd.)-supplemented Radio-Immunoprecipitation Assay (RIPA) buffer (Gibco; Thermo Fisher Scientific), the protein samples were loaded onto a sodium dodecyl sulfate polyacrylamide gel and subsequently blotted onto a PVDF membrane. Primary antibodies (Abs) were applied and incubated overnight in 5% bovine serum albumin at 4 °C. Western blotting analysis was conducted using ImageJ software (Bio-Rad Laboratories, Inc.). The primary Abs used were β-actin Ab (1:2000, 3700S, Cell Signaling Technology, Inc.), ATM Ab (1:2000, ab201022, Abcam), p-ATM Ab (1:1000, ab81292, Abcam), cleaved caspase-3 Ab (1:1500, ab32042, Abcam), cleaved caspase-9 Ab (1:1500, ab2324, Abcam), 53BP1 Ab (1:1000, ab175933, Abcam), H2AX Ab (1:1000, 7631S, Cell Signaling Technology), and p-H2AX Ab (1:500, 9718S, Cell Signaling Technology). For secondary Abs, the following were used: HRP-linked anti-mouse IgG (1:8000, 7076P2) and HRP-linked anti-rabbit IgG (1:5000, 7074P2), both from Cell Signaling Technology.
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