The Western blotting process was performed as previously described [28 (link)] and the PVDF membranes were incubated with the following primary antibodies at 4˚C overnight: rabbit anti-human CTBP1 (cat. no. 8684, Cell Signaling Technology), anti-human CTBP2 (cat. no. 13256, Cell Signaling Technology), anti-human phospho-Akt at Thr308 site (cat. no. 13038, Cell Signaling Technology), anti-human Akt (cat. no. 2920, Cell Signaling Technology), anti-human phospho-phosphatidylinositol-3-kinase p110α (cat. no. 4255, Cell Signaling Technology), and mouse anti-human β-actin (cat. no. ab 8227, Abcam). The density of each protein band was quantified using Image J software.
Mouse anti human β actin
Manufactured by Cell Signaling Technology
Sourced in United States
The Mouse anti-human β-actin is a primary antibody that specifically recognizes the β-actin protein, which is a widely expressed cytoskeletal protein. This antibody can be used to detect and quantify β-actin levels in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (5 mentions)
Chemiluminescence imaging system, by Bio-Rad (5 mentions)
Vimentin, by Cell Signaling Technology (4 mentions)
N cadherin, by Cell Signaling Technology (3 mentions)
Goat anti mouse igg secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Soluble recombinant human trail, by Enzo Life Sciences (2 mentions)
Rabbit anti human e cadherin, by Cell Signaling Technology (2 mentions)
Fscn1, by Abcam (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Snail, by Cell Signaling Technology (2 mentions)
Anti human akt, by Cell Signaling Technology (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Complete edta free protease inhibitor tablet, by Roche (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (1 mentions)
20 protocols using mouse anti human β actin
1
Western Blotting Analysis of Signaling Proteins
2
Colon Cancer Cell Line HCT-116 Protocol
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Colon cancer cell line, HCT-116, was received as a kind gift from Dr. Virupakshi Soppina (IIT Gandhinagar). DMEM, fetal bovine serum, pen-strep, trypsin-EDTA and cell extraction buffer were purchased from Invitrogen Corporation (Carlsbad, CA, USA). Complete EDTA-Free protease inhibitor tablets were purchased from Roche (Basel, Switzerland). Sodium dodecyl sulfate, tetramethylenediamine (TEMED), ammonium per sulphate, Tween-20, β-mercatopethanol, bromophenol blue, non-fat milk, and bovine serum albumin were purchased from Sigma-Aldrich (Darmstadt, Germany). Clarity ECL western blotting substrate and immunoblot PVDF western blotting membrane were purchased from Bio-Rad laboratories (Hercules, CA, USA). Rabbit anti-human Chk1 phospho Ser 317 (Cat. 12302), mouse anti-human phospho p70 S6 kinase Thr389 (Cat. 9206) were purchased from Cell Signaling Technology (Danvers, MA, USA), and mouse anti-human β-actin (Cat. SC47778) was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Anti-rabbit IgG HRP-linked secondary antibody (Cat. 7074) and anti-mouse IgG HRP-linked secondary antibody (Cat. 7076) were obtained from Cell Signaling Technology (Danvers, MA, USA). CellTiter-Glo® Luminiscent cell viability assay kit was purchased from Promega Corporation (Madison, WI, USA). 96- and 6-well plates were purchased from Corning (New York, NY, USA).
3
MMP16 Protein Expression Analysis
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Total proteins were extracted by RIPA lysis buffer(Roche, Indianapolis, IN, USA) and 1 mM PMSF on ice, proteins were separated by SDS-PAGE and then transmembrane. 5% skimmed milk was sealed at room temperature for 2 hours, and then incubated overnight at 4 °C with rabbit anti-human MMP16(1:500, Cell Signaling Technology, Danvers, MA, USA) and mouse anti-human β-actin(1:1000, Cell Signaling Technology, Danvers, MA, USA). TBST membrane was washed for 8 minutes and 5 times, the blots were incubated with horse radish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG for 2 hours. The TBST membrane was washed 5 times for 8 minutes each time.
4
Quantitative Western Blot Analysis
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RCC tissues and cells were lysed using Thermo Fisher Scientific RIPA buffer (Pierce; Thermo Fisher Scientific, Inc.). The Micro BCA protein assay kit was used to detect the protein concentration. Equal amounts of protein (50 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk (w/v) at room temperature for 1 h and subjected to incubation with the corresponding primary antibodies at 4°C overnight: Rabbit anti-human FoxM1 (1:1,000; cat. no. 5436), mouse anti-human MMP-2 (1:1,000; cat. no. 4022) and MMP-9 (1:1,000; cat. no. 3852) and rabbit anti-human PCNA (1:1,000; cat. no. 13110; all from Cell Signaling Technology, Danvers, MA, USA) and mouse anti-human β-actin (1:500; cat. no. sc-8432; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Subsequently, the PVDF membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000; cat. no. ab150077; Abcam, Cambridge, MA, USA) for 1 h. An enhanced chemiluminescence western blotting detection system (EMD Millipore) was used to detect the protein expression level. Immunodetection was visualized on a Gel Doc 2000 Imaging System (Bio-Rad Laboratories).
5
Evaluating TRAIL-induced Apoptosis Pathways
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Human recombinant interferon beta (IFNβ) was obtained from R&D System (NY, USA). Soluble, recombinant human TRAIL, Super FAS Ligand, the primary mouse monoclonal antibodies against TRAIL, TRAIL-R1 and TRAIL-R2 were purchased from Enzo Life Science (Paris, France). The caspase inhibitors Z-VAD-fmk, Z-IETD-fmk and Z-LEHD-fmk were obtained from R&D System (Wiesbaden, Germany). Antibodies used for immunoblotting were mouse anti-human β-actin (Cell Signaling, Danvers, MA, USA), mouse anti-human-TRAIL (Enzo Life Science), mouse anti-human caspase-3 (Cell Signaling), and mouse anti-human caspase-8 (Enzo Life Science). The goat anti-mouse IgG secondary antibody was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The FITC-active caspase-3 apoptosis test was obtained from BD Pharmingen (San Diego, CA, USA). Hoechst 33258 was purchased from Sigma (St. Louis, MO, USA), Rotitest Vital from Roth (Karlsruhe, Germany). Knock-down experiments for TRAIL-RNA were performed using TRAIL-siRNA (Dharmacon, Freiburg, Germany, Cat. Nr. L-011524-00-0010) and scrambled RNA (Dharmacon, Cat. Nr. D-001810-20).
6
Anticancer effects of natural compounds
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Lycorine (purity >98%) was obtained from Nanjing Purun Tech Co. and cisplatin (purity >98.5%) was obtained from Dalian Meilun Biotech. Curcumin (purity >98%) was purchased from Shanghai Jianglai Biotech and non-small cell lung cancer cell line A549 was from Nanjing Lishizi Biotech (Nanjing, China). Fetal bovine serum was purchased from Hyclone (Logan City, UT). DMEM were from Shanghai Feili i Biotech (Shanghai, China). Cell culture flasks, cell culture dishes, and 96- and 6-well plates were purchased from Tianjin Ruishina biotech (Tianjin, China). MTT and trypsin was bought from Nanjing Zhongshan Biotech (Nanjing, China). Wortmannin was purchased from Selleck (Houston, TX). Protein extraction kit and BCA protein quantification kit were from Shijiazhuang Haisen Chemical (Shijiazhuang, China). Mouse anti-human β-actin (Item No. 3700), rabbit anti-human AMPK (Item No 5831), rabbit anti-human mTOR (Item No. 2983), rabbit anti-human S6K (Item No. 2708), rabbit anti-human LC3 (Item No. 3868), rabbit anti-human caspase3 (Item No. 9662), rabbit anti-human phospho AMPK (Item No. 5256), and rabbit anti-human S6K (Item No. 9234) antibodies were bought from Cell Signaling Technology (Danvers, MA). Goat anti-rabbit IgG-HRP (sc-2004) and goat anti-mouse IgG-HRP (sc-2005) secondary antibodies were from Santa Cruz Biotechnology (Dallas, TX).
7
Western Blot Analysis of EMT Regulators
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Total protein was collected from cells by RIPA lysis buffer containing protease inhibitors (Roche, Indianapolis, IN, USA) and 1 mM PMSF on ice. Protein concentration was measured using the BCA-200 Protein Assay kit (Pierce, Rockford, IL, USA). After heat denaturation at 100°C for 5 min, proteins were separated by electrophoresis on 10% SDS–PAGE gels and then transferred onto nitrocellulose membranes (Pall Life Science, NY, USA). The membranes were blocked with 5% non-fat milk at room temperature for 1 h, and then incubated overnight at 4°C with rabbit anti-human E-cadherin(1:1000, Cell Signaling Technology, Danvers, MA, USA), N-cadherin(1:1000, Cell Signaling Technology, Danvers, MA, USA), vimentin(1:500, Cell Signaling Technology, Danvers, MA, USA), DNMT3A (1:500, Cell Signaling Technology, Danvers, MA, USA), DNMT3B(1:500, Cell Signaling Technology, Danvers, MA, USA), DNMT1(1:1000, Active Motif; Carlsbad, CA, USA), FSCN1(1:100000, Abcam, Cambridge, MA, USA), and mouse anti-human β-actin(1:1000, Cell Signaling Technology, Danvers, MA, USA). After washing with TBST, the blots were incubated with horse radish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG. Blots were visualized using ECL reagents (Pierce, Rockford, IL, USA) by a chemiluminescence imaging system (Bio-Rad, Richmond, CA, USA).
8
Western Blot Analysis of Phospho-PKCζ
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The cells were lysed in RIPA buffer (Heart Biological Technology Co., Ltd., Xi'an, China) containing 1% aprotinin, 1% activated Na3VO4 and 1% PMSF on ice. The protein concentration was detected according to the instructions of the Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Total proteins (50 µg) were loaded onto a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel, separated by electrophoresis and transfered onto PVDF membranes (Millipore, Boston, MA, USA). The membranes were blocked with 5% skim milk at room temperature for 2 h and then incubated with rabbit anti-human PKCζ (1:1,000, #sc-216; Santa Cruz Biotechnology, Inc., CA, USA), rabbit anti-human MMP2 (1:1,000, #10373-2-AP), MMP9 (1:500, #10375-2-AP), mouse anti-human β-actin (1:1,000, #60008-1-lg) (both from China Branch of Proteintech Co., Hubei, China) and rabbit anti-human phospho-PKCζ antibodies (1:500, #9378; Cell Signaling Technology, Boston, MA, USA) at 4°C overnight. The following day, the membranes were sequentially incubated with HRP-conjugated goat anti-rabbit IgG (1:3,000, #A0208) or HRP-conjugated goat anti-mouse IgG (1:3,000, #A0216) (Beyotime, Shanghai, China) at 37°C for 1 h. Immunoreactive proteins were detected using ECL reagent (Millipore) and the FluorChem FC system (Alpha Innotech, San Leandro, CA, USA). Bands were quantitated using ImageJ software.
9
Luteolin-Induced Cell Cycle Arrest and Apoptosis
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Luteolin was purchased from Sigma-Aldrich (EMD Millipore, Billerica, MA, USA); and was dissolved in dimethyl sulphoxide and its concentration was adjusted to 100 mmol/l, as a stock solution. The Cell Counting kit-8 was supplied by Beyotime Institute of Biotechnology (Haimen, China). Annexin V-fluorescein isothiocyanate (FITC) apoptosis and cell cycle detection kits were obtained from BD Biosciences (Franklin Lakes, NJ, USA). A bicinchoninic acid protein assay kit was purchased from Biosynthesis Biotechnology Co., Ltd. (Beijing, China) and monoclonal antibodies, including rabbit anti-human cell division cycle 2 (CDC2), cyclin-dependent kinase 2 (CDK2), cyclin B1, cyclin A, apoptotic protease activating factor 1 (APAF-1), cytochrome c, caspase-3, mouse anti-human procaspase-9, mouse anti-human caspase-9 and mouse anti-human β-actin, were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
10
Modulating Immune Signaling Pathways
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