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2 protocols using alexa fluor 594 anti mouse igg

1

Antibody Panel for Cellular Signaling

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Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236, mTOR, phospho-4EBP1, and GAPDH were from Cell Signaling, Phospho-FAK (Tyr397), HPDL from Thermo Fisher, Paxilin and LAMP2 from BD-bioscience; PAK1 from Proteintech. Secondary antibodies Alexa-fluor 594 anti-Rabbit IgG, Alexa-fluor 488 anti-Rabbit IgG, Alexa-fluor 594 anti-Mouse IgG and Alexa-fluor 488 anti-Mouse IgG were from Cell Signaling, IRDye 800CW and IRDye 680CW were from LI-COR. Alexa fluor TM 555 Phalloidin, Click-iT EdU Imaging Kits, CellEvent Caspase-3/7 Green Detection kit, NHS-Fluorescein, NHS-Alexa Fluor 555, pH-rodo iFL STP ester red and Hoechst 33342 were from Invitrogen. DRAQ5 was from LI-COR. Collagen I and Matrigel were from Corning. Geltrex was from Thermo Fisher. All media and dialyzed FBS were from Gibco, except for DMEM with no amino acid which was from US Biological life science and Plasmax which was kindly provided by Dr Tardito, CRUK Scotland Institute, Glasgow. The details of Plasmax composition were previously described by Vande Voorde and colleagues [18 (link)]. E64d (Aloxistatin) and PF573228 were from AdooQ Bioscience. GM6001 was from APEXBIO. Dynasore, Filipin, and EIPA were from Sigma. FRAX 597 was from bio-Techne. Nitisinone was from MCE. PP2 was from Generon. C13-tyrosine was from Sigma-Aldrich.
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2

Influenza Virus Infection in MDCK Cells

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MDCK cells were washed twice with PBS and incubated for 2 h with influenza virus (0.001 MOI) at 37 °C in 5% CO 2 atmosphere for adsorption. After 2 h, inoculum was decanted and infected cells were supplemented with maintenance medium with or without luteolin. After 18 h, MDCK cells were fixed for 10 min with 4% paraformaldehyde at room temperature, and washed three times with PBS to remove the fixation buffer. The cells were permeabilized in 0.5% Triton X-100 in PBS for 15 min. The samples were then incubated with 3% BSA for 1 h at room temperature and further incubated with the mouse anti-influenza virus M2 antibody overnight at 4 °C. After washing three times with TBST, cells were incubated with Alexa Fluor 488 anti-mouse IgG (TransGen) for 1 h. The nucleus was stained with Hoechst 33342 (Beyotime) for 5 min. Photos were taken with an Olympus TH4-200 microscope.
For co-localization studies with β-COP and GM130, fixed Vero cells were permeabilized in 0.5% Triton X-100 for 15 min and blocked with 3% BSA for 1 h at room temperature. The cells were incubated with rabbit anti-β-COP and mouse anti-GM130 antibody overnight at 4°C, rinsed with TBST, stained with Alexa Fluor 488 anti-rabbit IgG (TransGen) and Alexa Fluor 594 anti-mouse IgG (Cell Signaling Technology) for 1 h. The nuclei were stained with DAPI (ZSBIO, Beijing). Images were acquired with a Zeiss LSM 710 microscope.
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