Mouse monoclonal anti flag m2 antibody f1804

For indirect immunofluorescence staining, cells were fixed with 4% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, blocked with 1% BSA for 60 min, and then probed with the indicated primary antibodies, including mouse monoclonal anti-IAV NP antibody (1331, 1:200, ViroStat, Portland, ME, USA), rabbit anti-human galectin-3 antibody (sc-20157, 1:200, Santa Cruz), rabbit anti-influenza A PA antibody (GTX125932, 1:500, GeneTex), and mouse monoclonal anti-Flag M2 antibody (F1804, 1:500, Sigma-Aldrich), overnight at 4 °C. Alexa Flour 488-goat anti-mouse IgG (A-21202, 1:200, Invitrogen) and Alexa Flour 594-goat anti-rabbit IgG (A-11012, 1:200, Invitrogen) were used as secondary antibodies. The nuclei were counterstained with DAPI (Sigma-Aldrich). Images were acquired using an Olympus FV1000 confocal microscope or observed under fluorescence microscopy (Olympus, Tokyo, Japan).

Mammalian expression plasmid encoding human TMEM263 with a C-terminal epitope tag (Myc-DDK) was obtained from Origene (RC203933). Control pCDNA3.1 empty plasmid was obtained from Invitrogen. Rabbit polyclonal anti-human C12orf23 (TMEM263) was from Origene (TA333490). Mouse monoclonal JAK2 (C-10), STAT5 (A-9), growth hormone receptor (GHR) (B-12) were obtained from Santa Cruz (sc-390539, sc-74442, and sc-137184, respectively). Rabbit monoclonal antibody against phospho-JAK2 (Tyr1008)(D4A8) was obtained from Cell Signaling Technology (CST # 8082). Rabbit polyclonal antibody against phospho-STAT5 (Y694) was obtained from Cell Signaling Technology (CST#9351). Mouse monoclonal anti-FLAG M2 antibody (F1804) and mouse monoclonal anti-β-actin antibody (A1978) were obtained from Sigma. HRP-conjugated goat anti-rabbit (CST#7074) and goal anti-mouse (CST#7076) antibodies were from Cell Signaling Technology.

CGP57380 (C0993) was purchased from Sigma-Aldrich and used at 20 μM. Rapamycin (#9904), LY294002 (#9901), U0126 (#9903) and SB203580 (#5633) were purchased from Cell Signaling Technology (Danvers, MA, USA) and used at 100 nM, 20 μM, 10 μM, and 10 μM respectively. Mouse monoclonal antibodies against NDV phosphoprotein (P protein) and nucleocapsid protein (NP protein) were prepared in our laboratory. Puromycin and mouse monoclonal anti-Puromycin 12D10 (MABE343) were purchased from Merck Millipore (Billerica, MA, USA). Mouse monoclonal anti-FLAG M2 antibody (F1804) and anti-β-actin antibody (A1978) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and other primary antibodies were from Cell Signaling Technology. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or -mouse secondary antibody, tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse and fluorescein (FITC)-conjugated goat anti-rabbit secondary antibody were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Small interfering RNAs (siRNAs) designed specfically for human Mnk1 (sc-39106), eIF4E (sc-35284) and eIF4G (sc-35286) were from Santa Cruz Biotechnology (Dallas, TX, USA). RC DC Protein Assay kit (500–0199) was purchased from Bio-Rad.