Total cell protein was extracted on ice using RIPA lysis buffer in the presence of freshly added protease and phosphatase inhibitors (Thermo), then quantified by the BCA method (Pierce). A total of 30 μg protein extract per lane was loaded onto a 14% SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Pierce). Nonspecific binding was blocked with 5% nonfat milk in PBS. The membrane was incubated with rat anti-IL-33 (R&D) or rabbit anti-phospho-STAT6 (Cell signaling) antibody overnight at 4°C. IRDye 800CW goat anti-rabbit IgG or goat anti-rat IgG (LI-COR) was used as secondary antibody, and rabbit anti-GAPDH antibody (Abcam) was used as an internal standard.
Rabbit anti phospho stat6
Manufactured by Cell Signaling Technology
Rabbit anti-phospho-STAT6 is a primary antibody that specifically binds to the phosphorylated form of the STAT6 protein. STAT6 is a transcription factor that plays a key role in the signaling pathways activated by cytokines such as interleukin-4 (IL-4) and interleukin-13 (IL-13).
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx stain free gel, by Bio-Rad (2 mentions)
Mouse anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (2 mentions)
Image lab system, by Bio-Rad (2 mentions)
Trans blot turbo system, by Bio-Rad (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Rabbit anti relmα, by Thermo Fisher Scientific (2 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Rabbit anti gapdh antibody, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Rat anti il 33, by R&D Systems (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Facsdiva v9, by BD (1 mentions)
Mouse anti t gondii, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti akt, by Cell Signaling Technology (1 mentions)
Rabbit anti 4e bp1, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Hrp conjugated rabbit anti actin, by Cell Signaling Technology (1 mentions)
Rabbit anti stat6, by Cell Signaling Technology (1 mentions)
5 protocols using rabbit anti phospho stat6
1
Western Blot for IL-33 and STAT6
2
Western Blot Protein Analysis Protocol
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Samples were run on Bio-Rad Mini-PROTEAN TGX Stain-Free Gels, 4 to 15%, with a 10- or 12-well comb in tris/glycine/SDS buffer. Protein was transferred onto activated polyvinylidene difluoride membranes using the Bio-Rad Trans-Blot Turbo system according to the manufacturer’s instructions. Membranes were blocked with TBST (20 mM tris, 150 mM NaCl, and 0.05% Tween 20)/5% BSA for 1 hour and then incubated with primary antibodies in TBST/5% BSA with sodium azide at 4°C overnight. Primary antibodies included rabbit anti–phospho-STAT6 (Cell Signaling Technologies, 56554), rabbit anti-RELMα (PeproTech, 500-P214), and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, sc-32233). Membranes were washed three times with TBST and then incubated with horseradish peroxidase–conjugated secondary antibodies (anti-rabbit or anti-mouse) for 1 hour at room temperature. Membranes were washed three more times and then developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Protein was visualized using the Bio-Rad Image Lab system.
3
Analysis of T. gondii-Induced STAT6 Phosphorylation
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T25 flasks of confluent HFFs were infected with 1 million parasites for 2 h, after which the HFFs were dissociated and fixed with methanol for 10 minutes. The cells were stained with 1:200 rabbit anti-phospho-STAT6 (Cell Signaling #56554) and 1:200 mouse anti-T. gondii (Santa Cruz #SC-52255) overnight at 4°C, followed by 1:1000 goat anti-rabbit 488 (Invitrogen #A11008), 1:1000 goat anti-mouse 594 (Invitrogen #A11005), and 5 μg/mL DAPI for 1 h at room temperature. Data were collected on an LSR II flow cytometer (BD) running FACSDiva v9 (BD) and analysed with FlowJo v10 (www.flowjo.com ). The median anti-phospho-STAT6 signal in the infected cells was determined for each sample, and the median technical replicate taken to represent the biological replicate. The data were scaled to RHΔKU80 = 1 AU and differences between strains tested by two-sided Wilcoxon rank sum test with Benjamini-Hochberg adjustment.
4
Western Blot Analysis of Cellular Signaling Pathways
5
Western Blot Protein Analysis Protocol
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Samples were run on Bio-Rad Mini-PROTEAN TGX Stain-Free Gels, 4 to 15%, with a 10- or 12-well comb in tris/glycine/SDS buffer. Protein was transferred onto activated polyvinylidene difluoride membranes using the Bio-Rad Trans-Blot Turbo system according to the manufacturer’s instructions. Membranes were blocked with TBST (20 mM tris, 150 mM NaCl, and 0.05% Tween 20)/5% BSA for 1 hour and then incubated with primary antibodies in TBST/5% BSA with sodium azide at 4°C overnight. Primary antibodies included rabbit anti–phospho-STAT6 (Cell Signaling Technologies, 56554), rabbit anti-RELMα (PeproTech, 500-P214), and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, sc-32233). Membranes were washed three times with TBST and then incubated with horseradish peroxidase–conjugated secondary antibodies (anti-rabbit or anti-mouse) for 1 hour at room temperature. Membranes were washed three more times and then developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Protein was visualized using the Bio-Rad Image Lab system.
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