Total brain tissues were sonicated using a LabSonic homogenizer (B. Braun Biotech Inc., Allentown, PA, USA), and the protein concentration in the brain samples was then quantified using a bicinchoninic acid assay kit (Pierce, CA, USA). Samples were then analyzed by 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h with 5% milk containing 0.05% Tween in PBS. The blots were then incubated overnight at 4 °C with the following primary antibodies: β-actin (1:3000; Sigma-Aldrich), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-GRP78, mouse anti-CHOP, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-Akt, rabbit anti-phospho-4E-BP1, rabbit anti-phospho-p70 S6 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel). Membranes were then probed with secondary horseradish peroxidase-conjugated antibodies (1:3000; Amersham Biosciences, Little Chalfont, UK) for 1 h at room temperature. The blots were then visualized using chemiluminescent peroxidase substrate (ECL prime; GE Healthcare). Immunoreactivity for each protein band intensity was quantified using NIH ImageJ software [24 (link)] and normalized to β-actin as a loading control.
Secondary horseradish peroxidase conjugated antibodies
Manufactured by Cytiva
Sourced in United Kingdom
Secondary horseradish peroxidase-conjugated antibodies are laboratory reagents used in various immunoassay techniques. They consist of secondary antibodies that have been conjugated with the enzyme horseradish peroxidase. The enzyme can catalyze chromogenic or chemiluminescent reactions, allowing for the detection and quantification of target analytes in samples.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
Mouse anti chop, by Cell Signaling Technology (1 mentions)
Rabbit anti mtor, by Merck Group (1 mentions)
Rabbit anti grp78, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho p70 s6, by Cell Signaling Technology (1 mentions)
Rabbit anti akt, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho mtor ser2448, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho 4ebp1, by Cell Signaling Technology (1 mentions)
Mouse anti kca3, by Alomone (1 mentions)
Ps133 creb, by Cell Signaling Technology (1 mentions)
Mouse monoclonal antibody to β actin, by Merck Group (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Ps473 akt, by Cell Signaling Technology (1 mentions)
Total creb, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid protein assay kit, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Anti active β catenin, by Merck Group (1 mentions)
Anti egf receptor, by Cell Signaling Technology (1 mentions)
Chemiluminescent detection reagents ecl, by Cytiva (1 mentions)
6 protocols using secondary horseradish peroxidase conjugated antibodies
1
Western Blot Analysis of Brain Proteins
2
Prostate Cancer Cell Line Characterization
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C42, PC3, and LNCaP cell lines used in this study were previously described.5 , 17 22Rv1 prostate cancer cells were purchased at ATCC. Cell lines were tested for mycoplasma at Cell Engineering Shared Resource Laboratory WFUCCC.
Antibodies were obtained from the following sources: mouse monoclonal antibody to β‐actin from Sigma‐Aldrich (#A5316); secondary horseradish peroxidase–conjugated antibodies from Amersham Biosciences (NXA931). Antibodies to total CREB (#9197), pS133CREB (#9198), p157VASP (#84519), total VASP (#3132), pY202pT204ERK (#9101), total ERK (#4696), pS473AKT (#9271), and total AKT (#4685) were from Cell Signaling Technology.
Tissue culture reagents were purchased from Invitrogen. Epi, Epi bitartrate, and all other chemicals and reagents, unless otherwise specified, were purchased from Sigma‐Aldrich.
Antibodies were obtained from the following sources: mouse monoclonal antibody to β‐actin from Sigma‐Aldrich (#A5316); secondary horseradish peroxidase–conjugated antibodies from Amersham Biosciences (NXA931). Antibodies to total CREB (#9197), pS133CREB (#9198), p157VASP (#84519), total VASP (#3132), pY202pT204ERK (#9101), total ERK (#4696), pS473AKT (#9271), and total AKT (#4685) were from Cell Signaling Technology.
Tissue culture reagents were purchased from Invitrogen. Epi, Epi bitartrate, and all other chemicals and reagents, unless otherwise specified, were purchased from Sigma‐Aldrich.
3
Western Blot Analysis of Protein Lysates
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After various treatments, cell lysates were prepared in a lysis buffer containing 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, and 10 mg/ml leupeptin. Following centrifugation at 12,000 rpm for 10 min, the protein concentrations of the supernatants were determined with a bicinchoninic acid protein assay kit (Bio-Rad, Hercules, CA, USA). For Western blot analysis, equal amounts of cellular protein (30 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). These membranes were subsequently blocked overnight with 5% skim milk in tris-buffered saline containing 0.1% Tris-buffered saline tween 20 (TBST). After three washes with TBST, membranes were incubated with primary antibodies overnight at 4°C and then further incubated with secondary horseradish peroxidase-conjugated antibodies (Amersham Pharmacia Biotech, Buckinghamshire, UK). Expression levels were normalized to β-actin (sigma, St Louis, MO, USA). The image capture and analysis were performed using an enhanced chemiluminescence system (Pierce, Rockford, USA).
4
Western Blot Profiling of Cellular Pathways
5
Integrin, ILK, and TG2 Regulation
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Unless stated otherwise, chemicals and reagents were from Sigma–Aldrich. Monoclonal FN was from BD Biosciences; monoclonal TG2 (clone 4G3), integrin β1 (MAB1959; clone P5D2), integrin β1 (MAB2251; clone B3B11), and polyclonal p-ILK (Ser246) were from MilliporeSigma, and monoclonal TG2 (CUB 7402) and polyclonal TG2 (Ab-4) antibodies were from Thermo Fisher Scientific. Polyclonal ILK (clone 4G9), nonphospho (active) β-catenin at Ser33/37 and Thr41 (clone D13A1), p-FAKTyr576/577, FAK (clone D5O7U), GSK-3α/β, and p-GSK-3α/βSer21/9 (clone 37F11) antibodies were from Cell Signaling Technology; and GAPDH from Biodesign International. Secondary horseradish peroxidase–conjugated antibodies were from Amersham Biosciences and Santa Cruz Biotechnology, Inc. Lentiviral particles containing shRNA targeting TG2 and ILK and scrambled shRNA were purchased from Sigma–Aldrich. Recombinant human Wnt-3A was purchased from R&D Systems. ILK inhibitor, cpd-22, was purchased from MilliporeSigma.
6
Immunoblotting Antibody Protocols
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Anti-Cdk5 (C-8) (1:1000) antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas). Anticleaved Caspase-3 (Asp 175 ) (1:1000), anti-Caspase-3 (9662) (1:1000), anti-α-tubulin (2144) (1:1000) and anti-P35/25 (C64B10) (1:1000) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-TGF-β1, anti-TNF-α, anti-IL-1β and anti-insulin antibodies (1:500-1000) were obtained from Abcam (Cambridge, MA). Anti-β-tubulin (AC010) (1:5000) antibodies were obtained from ABclonal (Wuhan, China). Secondary horseradish peroxidase-conjugated antibodies (1:2000) were obtained from Amersham Biosciences (Piscataway, NJ). Secondary uorescence-conjugated Oregon Green and Texas Red antibodies (Molecular Probes, Eugene OR) were used after dilution at 1:400. Collagenase XI was purchased from Sigma. A rat/mouse insulin ELISA kit was obtained from Millipore (Billerica, MA).
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