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4 protocols using anti phosphotyrosine antibody

1

Inflammasome Activation Reagents and Antibodies

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LPS, isoamyl alcohol, 2-methylbutane, picrotoxin, 3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid, sodium orthovanadate, and monoclonal anti-mouse β-actin antibodies were all purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse ASC and caspase-1 antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX), anti-phosphotyrosine antibodies from Cell Signaling (Danvers, MA), anti-mouse and human IL-1β antibodies from R&D Systems (Minneapolis, MN), and anti-human caspase-1 antibodies from BIOMOL International LP (Plymouth Meeting, PA). Recombinant mature mouse IL-1β was also purchased from R&D Systems. Biotin conjugated anti- mouse and rabbit secondary antibodies were from GE Healthcare UK Limited (Little Chalfont, Buckinghamshire, UK) and anti-goat IgG from Jackson ImmunoResearch (West Grove, PA). For inflammasome stimulation, ATP, nigericin, alum Imject, apoSAA, poly (dA:dT), and anthrax lethal factor and protective antigen were purchased from Amersham Biosciences (Piscatawy, NJ), Invivogen (San Diego, CA), Thermo Scientific (Waltham, MA), PeproTech Inc. (Rocky Hill, NJ), Invivogen, and BEI Resources (Manassas, VA), respectively. Muscimol was acquired from MP Biomedicals (Santa Ana, CA), and ethanol from Pharmco AAPER (Brookfield, CT). ASC (Tyr144) phospho-specific antibody was purchased from ECM Biosciences (Versailles, KY).
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2

Immunoblotting and STAT3 Phosphorylation Analysis

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Cell extract, preparation and immunoblotting were performed as previously described.2 (link),51 (link) PYK2, STAT3 and JAK1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). AKT and pSTAT3 (pY 705), and anti-phosphotyrosine antibodies were from Cell Signaling (Boston, MA, USA). Anti-pJAK1 (pYpY 1022/1023) was from Invitrogen (Grand Island, NY, USA). pERK1/2 (pT 202/pY 204), ERK1 and β1 integrin antisera was from BD Biosciences (San Jose, CA, USA). pAKT (pS 473) antibody was from Imgenex (San Diego, CA, USA). Anti-pPYK2 (pY402) was from R&D Systems (Minneapolis, MN, USA). Actin antibody was from Sigma and horseradish peroxidase-conjugated secondary antibody was from Jackson ImmunoResearch (West Grove, PA, USA). STAT3 phosphorylation in primary cells was examined by FC. Cells were fixed in 1.6% paraformaldehyde, then permeabilized in 95% methanol for 10 min, and incubated in 1% fetal bovine serum overnight prior to staining with Alexa647-conjugated anti-pSTAT3 (pY 705; BD Pharmingen) for 2 h at 4 °C. Fibronectin was purchased from Invitrogen.
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3

KRAS Signaling Pathway Activation

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HEK 293T cells were transfected with siRNA via reverse transfection using Dharmafect 1 at a final concentration of 50 nm and seeded in regular 6-well–plates at a density of 1 × 106 cells/well. After 24 h incubation, cells were transiently transfected the Myc-tagged KRASG12D plasmid using the Lipo6000 Transfection Reagent (Beyotime, Shanghai, China, catalog number C0526) per the manufacturer's protocol and further cultured for 48 h. Cells were lysed in Pierce IP Lysis Buffer supplemented with protease and phosphatase inhibitors (Roche), and lysates were further incubated with 10 μg of anti-Myc tag antibody (agarose) (Abcam, Cambridge, England, UK, catalog number ab1253) or IgG-agarose beads (Abcam, catalog number ab104155) for 8 h at 4 °C. After washing the agarose beads three times with Pierce IP Lysis Buffer, the Myc-tagged-KRAS bound to agarose beads was released by the addition of SDS lysis buffer. Finally, samples were subjected to Western blotting analysis as previously described and blots were probed using an anti-phosphotyrosine antibody (Cell Signaling Technology, catalog number 9411, 1:1000) or anti-Myc antibody (Cell Signaling Technology, 1:1000).
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4

Western Blot Analysis of Cellular Proteins

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Cells were washed in PBS, scraped in SDS-PAGE loading buffer and boiled for 5 min. Proteins of total extracts were separated by SDS-PAGE and transferred to nitrocellulose membrane. Western blotting was carried out using standard methods. All antibodies were used at a dilution of 1:1000 of the stock in blocking buffer (5% skim milk prepared in TBS-Tween). The membranes were incubated with the indicated antibodies, washed with TBS-Tween, and signals were detected by using chemiluminescence. Antibodies to PKR and eIF2α were purchased from Santa Cruz Biotechnology. Antibodies to phospho-eIF2α (Ser-51) and anti-SUMO2 were purchased from Life Technologies. Anti-VSV-M antibody was from KeraFAST. Anti-HA monoclonal antibody was purchased from Covance. Anti-actin antibody was from MP Biomedicals. Anti-phosphotyrosine antibody was from Cell Signaling. Anti-VSV-G antibody was a generous gift of Dr. I Ventoso.
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