Mouse anti β actin mab

HEK 293T cells and Vero cells were grown in Dulbecco’s modified Eagle medium (DMEM) (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml of penicillin and streptomycin. CIK (Ctenopharyngodon idellus kidney) cells were grown in minimum essential medium (MEM) (Gibco-BRL) supplemented with 10% FBS respectively. GCRV-873 isolated and stored in the author’s laboratory was propagated in CIK cells as described previously [34 ].
GCRV VP1, VP4, VP6, VP7, NS38 and NS80 antibodies were generated and stored in our laboratory [31 (link), 35 (link)–38 (link)]. Mouse anti-Flag monoclonal antibody (mAb) was purchased from Abmart (Shanghai, China). Mouse monoclonal IgG2b isotype control antibody was purchased from eBioscience Inc. (San Diego, CA). Mouse anti-β-actin mAb, rabbit anti-poly-ubiquitin and anti-vimentin polyclonal antibodies (pAbs) were purchased from Proteintech (Wuhan, China). Alexa Fluor^® 488 or 568 donkey anti-rabbit IgG (H+L) antibody, Alexa Fluor^® 488 or 568 donkey anti-mouse IgG (H+L) antibody and Lipofectamine 2000 (Lipo2000) were purchased from Invitrogen Co. (Invitrogen, Carlsbad, USA).

Cells were cultured on coverslips with 70–80% confluence and were transfected by using Lipofectamin 2000 (Life Technologies). At 36 h post-transfection, cells were fixed with 4% paraformaldehyde in PBS (LEAGENE, Beijing, China) for 15 min, permeabilized with 0.1% Triton X-100 (GBCBIO Technologies Inc) for 5 min and blocked with 6% BSA (ZSGB-BIO, Beijing, China) in PBS, and incubated with Rhodamine Phalloidin (Cytoskeleton, Denver, CO, USA). To visualize different target molecules, slides were treated with mouse anti-β-actin mAb (Protein Tech), MitoTracker® Mitochondrion-Selective Probes (Life Technologies), Golgi-Tracker Red (Beyotime), ER-Tracker Red (Beyotime) or FITC Goat anti-mouse IgG (H+L) (Tianjin Sungene Biotech Co., Ltd). After being mounted with Prolong Gold Antifade Reagent with DAPI (Life Technologies), slides were observed with a Zeiss LSM710 confocal microscope (Zeiss, Oberkochen, Germany).