Gtx125932

HEK293-T cells were transfected with pCAGGs plasmids encoding for PA, PB2, PB1 (WT or mutant), 24 h p.t. the cells were lysed using ice-cold radio immunoprecipitation assay buffer (RIPA; 150 mM NaCl, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 25 mM TRIS (pH 7.5), 1% (v/v) Triton X-100, protease inhibitor cocktail). Clarified cell lysates were adjusted to equal amounts using Pierce™ BCA assay (Thermo Fisher), mixed with Laemmli buffer (4X), incubated at 95 °C for 5 min, and subjected to SDS-PAGE and western blot. Proteins were detected using the primary antibodies rabbit anti-PB1 (GTX125923, Genetex; 1:2000 in blocking buffer), rabbit anti-PA (GTX125932, Genetex; 1:1000 in blocking buffer), and mouse anti-Tubulin (clone DM1A, Sigma-Aldrich; 1:1000 in blocking buffer) and the secondary antibody anti-Rabbit IgG-800CW (LI-COR; 1:10,000 in blocking buffer), and anti-Mouse IgG-680RD (LI-COR; 1:10,000 in blocking buffer). Uncropped blots are provided in a source data file.

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).

HEK293S cells were lysed in low-salt lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Igepal (NP-40), and 5 mM EDTA at different times after infection. The samples were separated by SDS-PAGE (15%) and visualized by Western blotting with antibodies directed against M2e (MAb 37 or MAb 148, 1.3 μg/ml), RNP (polyclonal goat anti-RNP, 1/3,000, catalog no. NR-3133; Biodefense and Emerging Infections Resources Repository, NIAID, NIH), HA [polyclonal goat anti-influenza virus H1 (H0) hemagglutinin (HA) of A/Puerto Rico/8/1934 (H1N1), 1/1,000, catalog no. NR-3148; Biodefense and Emerging Infections Resources Repository, NIAID, NIH], PA (polyclonal rabbit, 1/1,000, catalog no. GTX125932; GeneTex), and PB2 (polyclonal rabbit, 1/1,000, catalog no. GTX125926; GeneTex), or GAPDH (polyclonal rabbit, 1/2,500, catalog no. ab9485; Abcam). For the detection, the following different DyLight-conjugated antibodies were used: goat IgG DyLight 680 conjugated (catalog no. 605-744-125; Rockland Immunochemicals, Inc.), goat anti-rabbit DyLight 800 conjugated (catalog no. 35571; Thermo Scientific), and goat anti-mouse DyLight 800 conjugated (catalog no. 35521; Thermo Scientific). Immunoreactive bands were detected using the Odyssey infrared imager (LI-COR Biosciences) and quantified using Image Studio Lite version 4.0.