Anti ha resin

P. aeruginosa strains containing plasmid-borne NrnC_Bh-HA were grown overnight, followed by dilution to an OD₆₀₀ = 0.1 in fresh LB supplemented with 60 µg/ml gentamicin. Cultures were allowed to grow to an OD₆₀₀ = 0.8. Arabinose was added to a final concentration of 0.2% to induce protein expression for 2 hr at 37°C. Following induction, cultures were normalized by OD, pelleted, and flash frozen in liquid nitrogen. Cells were resuspended in lysis buffer (150 mM NaCl, 25 mM Tris-Cl pH 7.5) followed by sonication. Anti-HA resin (Sigma) was prewashed with lysis buffer. Resin was added to the cleared lysate and incubated with rotation for 1 hr at 4°C. Following binding, the HA resin was washed with lysis buffer, boiled in SDS loading buffer, and resolved by SDS-PAGE. Western blot transfer to a PVDF membrane proceeded for 90 min at constant 0.25 A, followed by overnight blocking with superblock (ThermoFisher) at 4°C. Rabbit anti-HA primary antibody (Takara Bio) was diluted to 1:100 in TBS-T and incubated with the membrane for 1 hr at 20°C. Following washes with TBS-T, an HRP-conjugated, anti-rabbit antibody (GE Life Sciences) was diluted to 1:5000 in TBS-T and incubated with the membrane for 30 min at 20°C. The membrane was washed with TBS-T before treating with SuperSignal West Femto (ThermoFisher) ECL reagent, followed by imaging with a Bio-Rad Chemidoc system.

HEK293T cells were transfected with 3×Flag_D1ICD in pcDNA3.1. Transfected cells or NCSCs (±3×HA_D1ICD_Flag) were treated with IP lysis buffer (Thermo Fisher Scientific) with proteasome inhibitor cocktail (Nacalai Tesque) and immunoprecipitated with anti-Flag M2 resin or anti-HA resin (Sigma), respectively. The proteins were eluted with 3×Flag peptide (Sigma) or SDS-PAGE sample buffer (Nacalai Tesque).

The experiment of tandem affinity purification–mass spectrometry (TAP–MS) was performed according to the methods of Puig et al. (2001) (link). The WT strain, PoDot1-TAP, and positive control strain were cultured in fermentation medium with 1% cellulose plus 1% wheat bran as carbon sources at 30°C, 200 rpm for 60 h. About 40 g of mycelium for each sample was collected by vacuum filtration and liquid nitrogen grinding. Then, via the tandem affinity purification by Ezview^TM Red ANTI-FLAG M2 Affinity resin (Sigma–Aldrich, United States) and ANTI-HA resin (Sigma–Aldrich, United States), the eluent containing PoDot1 and interacted proteins was obtained. The two-step eluent was analyzed by silver staining of SDS-PAGE, Western blot, and mass spectrometry to determine the presence of the bait protein PoDot1 and interacted proteins of PoDot1. The mass spectrometry data were analyzed using the MASCOT engine (Matrix Science, United Kingdom; Version 2.2) for a non-redundant international protein index from the European Bioinformatics Institute. Three biological repeats were set for each strain. Proteins that were unique to PoDot1-TAP eluent and with unique pep count ≥ 3 were selected for further bioinformatics analysis.