Western blot assay was performed to detect the expressions of ZHX1, Bax, and Bcl-2. Cells or tissues were lysed with radio immunoprecipitation assay buffer (RIPA, Beyotime, China) containing protease inhibitors (Roche, Complete Mini). The proteins (30 μg) were subjected to 8%–10% SDS-polyacrylamide gel electrophoresis and transferred onto Hybond ECL membranes (Amersham). The membranes were incubated for 1 h at room temperature (RT) in blocking buffer (5% skim milk in TBS-T) and then incubated with the appropriate antibodies (ZHX1 antibody [Cat. No: 13903-1-AP, Proteintech, 1:500]; Bax [Cat. No: 2772s, CST, 1:1000]; Bcl-2 [Cat. No: 2870s, CST, 1:1000]) and β-actin antibody from Abcam overnight at 4°C. After washing with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody (1:10,000 dilution; Sigma) for 2 h at room temperature. Detection was performed using western blot detection reagents (Odyssey).
Horseradish peroxidase conjugated anti rabbit or anti mouse antibody
Manufactured by Merck Group
Sourced in United States
Horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody is a laboratory reagent used for detection and quantification of target proteins in various immunoassays. It consists of a secondary antibody that binds to the primary antibody directed against the target, with a horseradish peroxidase enzyme attached for signal amplification.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Bovogen (3 mentions)
Fitc conjugated anti mouse igg antibody, by Merck Group (3 mentions)
Trtic conjugated anti rabbit igg antibody, by Merck Group (3 mentions)
Hybond ecl membrane, by Cytiva (2 mentions)
Sunitinib malate su 11248, by Merck Group (2 mentions)
Pdcd4 d29c6 xp anti rabbit monoclonal antibody, by Cell Signaling Technology (2 mentions)
β actin antibody, by Abcam (1 mentions)
Complete mini, by Roche (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Glial fibrillary acidic protein (gfap), by Proteintech (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
β actin, by Proteintech (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Bcl2 associated x (bax), by ABclonal (1 mentions)
Bcl 2, by ABclonal (1 mentions)
Caspase 8, by ABclonal (1 mentions)
Caspase 3, by ABclonal (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Neratinib, by Selleck Chemicals (1 mentions)
Pelitinib, by Selleck Chemicals (1 mentions)
6 protocols using horseradish peroxidase conjugated anti rabbit or anti mouse antibody
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot Analysis of Astrocyte Proteins
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Proteins from astrocytes were separated by 12% SDS-PAGE after treatment, and samples were analyzed by western blotting. Proteins in the gels were transferred to nitrocellulose membranes using a semidry transfer unit at 0.04 mA for 50 min. The membranes were washed with TBS/T three times and then with a blocking buffer. The membranes were incubated overnight in antibodies against GFAP (Proteintech, USA), Bax (ABclonal, USA), Bid (ABclonal, USA), Caspase-3 (ABclonal, USA), Caspase-8 (ABclonal, USA), Bcl-2 (ABclonal, USA), p53 (ABclonal, USA), and β-actin (Proteintech, USA). The membranes were washed with TBS/T three times and then incubated with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody (Sigma–Aldrich, USA). Immunoreactive bands were detected using ECL reagents (EMD Millipore, USA) and captured by a ChemiDoc Imaging System (Bio-Rad, USA). ImageJ software analysis was employed to measure the image densitometry of target protein bands.
3
Western Blot Analysis of FoxO3a
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Total proteins were extracted from the cells in cell lysis buffer (50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% NP-40, 1 mM PMSF), and the protein concentration was determined by the BCA method. The proteins (30 μg) were subjected to 8-10% SDS-polyacrylamide gel electrophoresis and transferred onto Hybond ECL membranes (Amersham). The membranes were incubated for 1 h at room temperature in blocking buffer (5% skimmed milk in TBS-T) and then incubated with the appropriate antibodies (rabbit anti-FoxO3a (cat. no. 12829, Cell Signalling Technology; 1 : 1000) and mouse anti-β-actin (Millipore; 1 : 10,000)) overnight at 4°C. After being washed with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody (1 : 10,000; Sigma) for 2 h at room temperature. Western blot detection reagents (Odyssey) were used to detect proteins on the membranes. The corresponding bands were semi-quantitatively analysed by ImageJ (NIH, version 1.8.0).
4
Comparative Analysis of Tyrosine Kinase Inhibitors
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Total 11 TKIs were tested in this study. DMSO was used as a solvent for the TKIs and also as the negative vehicle control. Pyrimethamine 5 μM was used for positive control. Afatinib (BIBW2992), AG1478, dacomitinib (PF299), erlotinib (OSI-420), gefitinib (ZD1839), lapatinib, neratinib, AZD9291 (osimertinib), pelitinib, and nintedanib (BIBF 1120) were purchased from Selleck Chemicals (Houston, Texas, USA). Dimethyl sulfoxide (DMSO), pyrimethamine, and sunitinib malate (SU 11248) were purchased from Sigma Aldrich (St. Louis, Missouri, USA).
Bovine serum albumin was purchased from Bovogen Biologicals (Melbourne, Australia). Antibodies against β-actin were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). FITC-conjugated anti-mouse IgG antibody, TRTIC-conjugated anti-rabbit IgG antibody, and horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies were purchased from Sigma Aldrich. Mouse Tg563 monoclonal antibody was cloned in our laboratory. PDCD4 (D29C6) XP® anti-rabbit monoclonal antibody was purchased from Cell Signaling.
Bovine serum albumin was purchased from Bovogen Biologicals (Melbourne, Australia). Antibodies against β-actin were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). FITC-conjugated anti-mouse IgG antibody, TRTIC-conjugated anti-rabbit IgG antibody, and horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies were purchased from Sigma Aldrich. Mouse Tg563 monoclonal antibody was cloned in our laboratory. PDCD4 (D29C6) XP® anti-rabbit monoclonal antibody was purchased from Cell Signaling.
5
Antibody Acquisition and Characterization
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Afatinib (BIBW2992) and Erlotinib (OSI-420) were purchased from Selleck Chemicals (Houston, Texas, USA). Dimethyl sulfoxide (DMSO), pyrimethamine, and Sunitinib malate (SU 11248) were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Bovine serum albumin was purchased from Bovogen Biologicals (Melbourne, Australia).
Antibodies against Phospho-STAT1 (Tyr701) (58D6), Phospho-STAT2 (Tyr690), Phospho-STAT3 (Ser727), Phospho-STAT5 (Tyr694) (C11C5), Phospho-STAT6 (Tyr641), and β-actin were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). Antibodies against STAT1 p91(c-111), STAT6 (M-20), Phospho-JAK1 (Tyr1022/Tyr1023), and Phospho-JAK3 (Tyr980) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). FITC-conjugated anti-mouse IgG antibody, TRTIC-conjugated anti-rabbit IgG antibody, and horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies were purchased from Sigma Aldrich. Mouse Tg563 monoclonal antibody was cloned in our laboratory.
Antibodies against Phospho-STAT1 (Tyr701) (58D6), Phospho-STAT2 (Tyr690), Phospho-STAT3 (Ser727), Phospho-STAT5 (Tyr694) (C11C5), Phospho-STAT6 (Tyr641), and β-actin were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). Antibodies against STAT1 p91(c-111), STAT6 (M-20), Phospho-JAK1 (Tyr1022/Tyr1023), and Phospho-JAK3 (Tyr980) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). FITC-conjugated anti-mouse IgG antibody, TRTIC-conjugated anti-rabbit IgG antibody, and horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies were purchased from Sigma Aldrich. Mouse Tg563 monoclonal antibody was cloned in our laboratory.
6
Afatinib Inhibits Toxoplasma gondii Growth
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Afatinib (BIBW2992) was purchased from Selleck Chemicals (Houston, Texas, USA) as a TKI. DMSO and pyrimethamine were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Afatinib was treated with concentration of 5 mM which obtained over 98% of inhibition of T. gondii growth within the host cells [18 (link)]. Bovine serum albumin was purchased from Bovogen Biologicals (Melbourne, Australia). FITC-conjugated anti-mouse IgG antibody, TRTIC-conjugated anti-rabbit IgG antibody, and horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies were purchased from Sigma Aldrich. Antibody against β-actin was purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). Mouse Tg563 monoclonal antibody was cloned in our laboratory. PDCD4 (D29C6) XP® anti-rabbit monoclonal antibody was purchased from Cell Signaling (Boston, Massachusetts, USA) Chemicals for the analytical uses as iodoacetamide was purchased from Sigma-Aldrich. Tris-HCl and urea were obtained from Merck (Branchburg, New Jersey, USA). All other chemicals were acquired from standard sources and were of molecular biology grade or higher.
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