Monoclonal rabbit anti ha

HEK293T cells and Hela cells were transfected with plasmids for 24–36 h. Cells were fixed in 4% paraformaldehyde at room temperature for 15 min. After being washed three times with PBS, permeabilized with PBS containing 0.1% Triton X-100 for 5 min, washed three times with PBS, and finally blocked with PBS containing 5% BSA for 1 h. The cells were then incubated with the monoclonal mouse anti-Flag antibody (F3165, Sigma) and Monoclonal rabbit anti-HA (H6908, Sigma) overnight at 4°C, followed by incubation with FITC-conjugate donkey anti-mouse IgG (Abbkine) and Dylight 649-conjugate donkey anti-rabbit IgG (Abbkine) for 1 h. After washing three times, cells were incubated with DAPI solution for 5 min, and then washed three more times with PBS. Finally, the cells were analyzed using a confocal laser scanning microscope (Fluo View FV1000; Olympus, Tokyo, Japan).

FlAsH-EDT₂ was purchased from Toronto research chemicals Inc., and the transfection reagents Lipofectamine^TM 2000 were from Invitrogen. The monoclonal mouse anti-FLAG antibody was from Abcam and the monoclonal rabbit anti-HA was from Sigma. Pierce^® Classic IP Kit (26146) was from Thermo Scientific. KOD-Plus-Mutagenesis Kit was from TOYOBO. The plasmid purification kit (12362), nickel nitrilotriacetic acid (Ni-NTA) resin and Sephadex G-25 resin were purchased from QIAGEN. DEAE Sepharose^TM Fast Flow was purchased from GE Healthcare Bio-Science. SH-SY5Y cells were from the National Laboratory of Neurobiology of Fudan University. All other reagents were of analytic grade.

For IF, cells were fixed and incubated with antibodies as described in our previous study^{20 (link)}. The following dye-conjugated secondary antibodies were used for this analysis: Alexa Fluor 647 donkey anti-goat IgG H + L, Alexa Fluor 488 donkey anti-rabbit IgG H + L, and Alexa Fluor 594 donkey anti-mouse IgG H + L (Life Technologies). Cells were examined on a Leica confocal microscope.
For WB, samples were also analyzed as described in our previous study^{20 (link)} and detected on a Fujifilm LAS-4000 imaging system. The indicated primary and horseradish peroxidase-conjugated secondary antibodies (ThermoFisher Scientific) were used.
The primary antibodies goat polyclonal anti-TIA-1 (Cat#sc-1751) and mouse monoclonal anti-GAPDH (Cat#sc-32233) were purchased from Santa Cruz Biotechnology. Mouse monoclonal anti-G3BP (Cat#611127) was purchased from BD Transduction Laboratories. Rabbit polyclonal anti-eIF4E (Cat#A2162) was purchased from ABclonal. Rabbit monoclonal anti-eIF4GI (Cat#2858 S) and rabbit monoclonal anti-DYKDDDDK (Flag; Cat#14793 S) were purchased from Cell Signaling Technology. Mouse monoclonal anti-Flag (Cat#F1804), mouse monoclonal anti-HA (Cat#H9658), and rabbit monoclonal anti-HA (Cat#H6908) were purchased from Sigma-Aldrich. Mouse monoclonal anti-VP1 (Clone 22 A14) was purchased from Abmax^{49 (link)}.