Rat anti ha tag

Late trophozoite parasites cultured with or without aTc were isolated using a 65% Percoll gradient. Recovered parasites were resuspended in Laemmli Sample Buffer (Bio-rad) and β-mercaptoethanol (Sigma-Aldrich) and loaded on a 10% Mini-PROTEAN® TGX™ Precast Protein Gel. Samples were transferred to 0.2 μm polyvinylidene difluoride (PVDF) membrane using Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was blocked in 5% skim milk in 0.1% PBS-Tween (PBS-T) for 1 h at room temperature and incubated overnight with rat anti-HA tag (Roche) and mouse anti-Actin (Invitrogen) at 1:3000 in 2% Bovine Serum Albumin (BSA). The blot was then washed 3 times in PBS-T and probed with goat anti-rat HRP (Biolegend) and goat anti-mouse HRP (Biolegend) at 1:10,000 in 2% BSA PBS-T for 1 h at room temperature. The blot was imaged on ChemiDoc MP (Bio-Rad) in Clarity Max Western ECL Substrate (Bio-Rad). Western blot analysis was done using Image Lab v6.0 (Bio-Rad).

Cells grown and transfected on 15×15 mm cover slips were fixed with 4% PFA, permeabilized with Triton-X 100 and stained with rat anti-HA-Tag (Roche), mouse anti-CD63 (LAMP3) (Chemicon) or goat anti-calnexin (Santa Cruz) primary antibodies. Alexa Fluor® 488 donkey anti-rat IgG (H + L), Alexa Fluor® 555 donkey anti-mouse IgG (H + L) and Alexa Fluor® 555 F(ab')₂ fragment of goat anti-mouse or anti-goat IgG (H + L) secondary antibodies (Invitrogen) were used to visualize the signal. Coverslips were viewed with a Zeiss Axiovert 135 microscope and images were recorded with a Zeiss AxioCam MR camera and the Axiovision 3.1 software.

A549 cells were seeded on glass coverslips and transfected with pCAGGs plasmids expressing either WT or mutants of PB2, PB1 (in combination with nHA-PA), and PA (in combination with nHA-PB1)^{74 (link)} using XtremeGene^TM (Roche) 24 h p.t., cells were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 3% BSA in PBS (blocking buffer). For immunostaining coverslips were incubated with the primary antibodies rabbit anti-PB2 (GTX125926; Genetex; 1:3000 in blocking buffer), rabbit anti-PB1 (GTX125923, Genetex; 1:3000 in blocking buffer), rabbit anti-PA (GTX125932, Genetex; 1:3000 in blocking buffer), rat anti-HA-Tag (clone 3F10, Roche; 1:500 in blocking buffer), overnight at 4 °C and with secondary antibodies anti-rabbit Alexa Fluor 488 (Invitrogen; 1:2000 in blocking buffer) or anti-rabbit Alexa Fluor 568 (Invitrogen; 1:2000 in blocking buffer) and anti-rat Alexa Fluor 488 (Invitrogen; 1:2000 in blocking buffer) for 1 h at room temperature. Cell nuclei were stained with DAPI (Thermo Fisher Scientific) for 20 min at room temperature. Coverslips were mounted using Mounting Medium S3023 (Dako Omnis) and examined using an LSM-800 Airyscan confocal microscope (Carl Zeiss) and ZEN software (v2.6).