Ab3792

Similar to what we previously described^{12 (link)}, the Flag-tagged human TIPRL (Flag-TIPRL), V5-tagged human PP2Ac α isoform (V5-PP2Ac), and their mutants were cloned into murine retroviral vectors bearing a cytomegalovirus promoter. After co-transfection into 293T cells (Thermal Scientific), the transfection and overexpression efficiency of Flag-TIPRL and V5-PP2Ac were monitored by western blot using antibodies that specifically recognize Flag-tag (Sigma, M2, 1:1000), V5-tag (Millipore, AB3792, 1:1000), or PP2Ac (Millipore, 1D6, 1:1000). The interaction between WT or mutant Flag-TIPRL and WT V5-PP2Ac was determined by co-immunoprecipitation using anti-FLAG affinity gel (Sigma, A2220), and the level of Flag-TIPRL and the associated PP2Ac were determined by western blot using antibodies mentioned above. The same method was applied to study the interaction between Flag-TIPRL and WT or mutant V5-PP2Ac. The original images of western blot are shown in supplementary Fig. 14.

FLAG- or V5-tagged B′ substrates (FLAG-SENP6 (Addgene plasmid #18065), FLAG-USP53 (Addgene plasmid #22606), FLAG-GLI2 (Addgene plasmid #84920) and V5-CIP2A) and YFP-B′γ1 were co-transfected into 293T cells and cultured by similar procedures mentioned above. The transfection and overexpression efficiency of both proteins were monitored by western blot using antibodies that specifically recognize YFP-tag, FLAG-tag (M2, Sigma-Aldrich, St Louis, MO, USA) and V5-tag (Millipore, AB3792). The interaction between tagged-B′γ1 substrates and YFP-B′γ1 were recognized by co-immunoprecipitation using anti-FLAG and anti-V5 antibody immobilized on protein G magnetic beads (Invitrogen) before immunoprecipitate YFP-B′γ1 48 h after transfection. Cells were lysed in lysis buffer (50 mm Tris-HCl pH 8.0, 150 mm NaCl, 1 mm EDTA, 1 mm Dithiothreitol and 0.5% Triton X-100) and 400 μg of cell extracts were immunoprecipitated at 4 °C in lysis buffer for 8 h followed by western blot. A unit of 80 μg of whole-cell extracts were examined by western blot to examine the protein expression. The experiments were repeated three times and the representative results were shown.

Cells were washed twice with 1× PBS and total proteins were directly extracted in RIPA buffer (50 mM Tris-HCl pH 7.4, 50 mM NaCl, 2 mM EDTA, 0.1% SDS, 1× PhosSTOP™, 1× cOmplete™ EDTA free protease inhibitor cocktail (Roche)). A total of 20 μg of whole-cell proteins were separated on a NuPAGE™ 4–12% Bis-Tris Protein Gels and transferred on a nitrocellulose membrane using Trans-Blot® Turbo^TM Blotting System. Membranes were saturated with 5% milk and incubated overnight at 4 °C with the primary antibodies against RELA (sc-109, Santa Cruz, 1:1000), Tax (1A3, Covalab, 1:500), DDX17 (ab24601, Abcam, 1:2000), DDX5 (ab10261, Abcam, 1:2000), actin (sc-1616, Santa Cruz, 1:1000), GAPDH (sc-32233, Santa Cruz, 1:10,000), H3 (ab1791, Abcam, 1:10,000), V5 (AB3792, Millipore, 1:1000), and α-Tubulin (sc-32293, Santa Cruz, 1:2000). After three washes with 1× TBS-Tween, membranes were incubated 1 h at room temperature with the secondary antibodies conjugated with the HRP enzyme and washed three times as above. Finally, the HRP substrate (GE Healthcare or Immobilon Forte (Millipore)) was applied to the membrane for 5 min, and the chemiluminescence was read on Chemidoc (BioRad).

The Duolink^® Proximity Ligation Assay (PLA) (Sigma-Aldrich) fluorescence method was applied to detect in situ protein–protein interaction following the manufacturer’s instructions. Third instar larval eye discs expressing proteins of interests were used in this assay. Rabbit anti-V5 (1:1,000, AB3792, Millipore) and mouse anti-GFP (1:500, 4C9, DSHB) antibodies were used to assess interactions between TER94 and Mu2. Rabbit anti-Myc (1:1,000, GTX109636, GeneTex) and mouse anti-GFP (1:500, 4C9, DSHB) antibodies were used to assess interactions between p53 and Mu2. Images were acquired with a Zeiss LSM 800 confocal microscope.

HEK 293T cells, routinely tested to exclude mycoplasma contamination, were transiently transfected, using the calcium phosphate method^{63 (link)}. For co-immunoprecipitation, cells were lysed in 1% Triton-X, 20 mM Tris, pH7.5, 50 mM NaCl, 50 mM NaF, 15 mM Na₄P₂O₇, 0.1 mM EDTA, 2 mM Na₃VO₄ supplemented with protease inhibitor mix (Roche), incubated with 30 µl anti-Flag M2-agarose beads (A2220; Sigma-Aldrich, Eschenstrasse 5, 82024 Taufkirchen, Germany) for 2 h, and washed with lysis buffer. Precipitates were fractionated by SDS/PAGE, and analyzed by mouse monoclonal Flag antibody (1:3000) (F3165; Sigma-Aldrich) and rabbit anti-V5 (1:4000) (AB3792, Sigma-Aldrich). Uncropped Western blots are shown in Supplementary Fig. 13.

The mouse monoclonal antibodies (mAbs) against the PB2, PB1, PA, and NP proteins of IAV were generated in our laboratory [29 (link)]. The following primary antibodies were obtained from commercial sources: the rabbit anti-HACD3 polyclonal antibody (pAb) (28572-1-AP; Proteintech, Wuhan, China), rabbit anti-SQSTM1/p62 pAb (18420-1-AP; Proteintech), rabbit anti-Myc pAb (A00172; Genscript, Nanjing, China), mouse anti-Myc mAb (A00704; Genscript), rabbit anti-GST pAb (A00097; Genscript), rabbit anti-V5 pAb (AB3792; Sigma-Aldrich, Saint Louis, MO, USA), mouse anti-V5 mAb (V8012; Sigma-Aldrich), mouse anti-GAPDH mAb (60004-1-Ig; Proteintech), rabbit anti-HA pAb (11692-T62; Sino Biological, Beijing, China), rabbit anti-NA pAb (GTX125974; Genetex, Irvine, CA, USA), rabbit anti-M1 pAb (GTX125928; Genetex), rabbit anti-NS1 pAb (GTX125990; Genetex). The secondary antibodies, DyLight 800 goat anti-mouse IgG (H+L) (072-07-18-06; KPL, Gaithersburg, MD, USA) and DyLight 800 goat anti-rabbit IgG (H+L) (072-07-15-06; KPL), were used for Western blotting. The secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG (H+L) (A-11034; Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 633 goat anti-mouse IgG (H+L) (A-21052; Invitrogen), were used for confocal microscopy.