Anti mouse secondary antibody

For crude protein extracts, cells were lysed in RIPA buffer containing 1% NP40 and 0.1% SDS (Carl Roth) supplemented with ‘Complete’ and ‘PhosStop’ protease/phosphatase inhibitor cocktail (Sigma Aldrich) as described^{50 (link)}. Immunodetection of cellular extracts was performed using an anti-KIBRA (Santa Cruz Biotechnology; 1:500), anti-SP1 (Merck; 1:1000), anti-YAP (Santa Cruz Biotechnology; 1:1000), anti-pYAP (Ser127; Cell Signaling; 1:1000), anti-LATS1 (Merck; 1:1000), anti-pLATS1 (Thr1079; Cell Signaling; 1:500) and anti-rabbit secondary antibody (Santa Cruz Biotechnology; 1:20000 or Merck; 1:10000). Sample loading was controlled by β-actin detection (Cell Signaling; 1:5000) and anti-rabbit secondary antibody (Santa Cruz Biotechnology; 1:10000 or Merck; 1:20000). ZFP226 detection was conducted using anti-HA antibody (Cell Signaling; 1:1000) and anti-mouse secondary antibody (Santa Cruz Biotechnology; 1:20000). Western blots were repeated at least three times and band intensities were quantified using ImageJ^{51 (link)}.

Western blotting was carried out by transfering whole cell protein from 10% SDS‐PAGE onto the PVDF membranes (Millipore) and incubating the membranes with the primary and secondary antibodies. Primary antibodies against PRKD3, p‐ERK1/2 (Thr202/Tyr204), ERK1/2, p‐c‐MYC (Ser62) and c‐MYC were purchased from Cell Signalling Technology. β‐actin primary antibody, Anti‐rabbit and antimouse secondary antibody were purchased from Santa Cruz Biotechnology.

Salts were purchased from Sigma-Aldrich (Milan, Italy). Reagents were obtained from Mallinckrodt Baker (Milan, Italy). The Sartorius Vivaflow 200 ultrafiltration system, equipped with a 10 kDa cutoff polythersulfone membrane, was obtained from Sartorius (Florence, Italy). Pierce Concentrators were from Thermo Fisher Scientific (Milan, Italy). Anion exchange chromatography was performed on an FPLC Akta Basic equipped with a UV-Vis detector (GE-Healthcare, Milan, Italy) using a HiTrap DEAE FF column with a volume of 5 mL (GE-Healthcare, Milan, Italy). Gel filtration chromatography was conducted on an HPLC Akta Basic equipped with a UV-Vis detector (GE Healthcare, Milan, Italy) using a progel-TSK G2000 SWXL column 30 cm × 7.8 mm (Supelco, Merck KGaA, Darmstadt, Germany). Dialysis was performed using Spectra/Por 3.5 kDa MWCO membranes (Spectrum Labs, Fisher Scientific Italia, Rodano, Milan). PVDF membranes for western blotting were obtained from Millipore (Milan, Italy). The monoclonal antibody mAbKT4 was used to detect the KT [9 (link)]. The anti-mouse secondary antibody was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The ECL (enhanced chemiluminescence) system used for the immunodetection was obtained from Amersham Pharmacia Biotech (Milan, Italy).

Briefly, 1.2 × 10⁶ K562 cells were pelleted and resuspended in 100 µl hot lysis buffer (1% SDS, 10 mM Tris-HCl, pH 8.0) supplemented with 1X Halt™ protease and phosphatase inhibitor cocktail (Thermo Scientific, catalog # 78446). Samples were sonicated 20 times with 3 s ON and 3 s OFF cycles. Protein extracts were diluted in Laemmli buffer and heated at 70 °C for 10 min before loading on a 7.5% polyacrylamide Mini-PROTEAN TGX Stain-Free™ gel (BioRad, catalog # 4568023). Nitrocellulose membranes were blocked with 5% milk dissolved in PBST and incubated overnight at 4 °C using an anti-α-Na⁺/K⁺ ATPase antibody (Invitrogen, catalog # MA3-928) diluted 1:1000 in PBST 5% milk, and 1 h at room temperature using an anti-mouse secondary antibody (Cell Signaling Technology, catalog # 7076 S) diluted 1:5000 in PBST 5% milk. Membranes were subsequently incubated for 1 h at room temperature with an anti-α-Tubulin antibody (Santa Cruz Biotechnology, catalog # sc-32293) diluted 1:3000, followed by 1 h at room temperature using the same anti-mouse secondary antibody. The uncropped scans of all blots from this study are provided in Supplementary Fig. 26.

To confirm that candidate effector proteins had been produced in N. benthamiana and N. tabacum leaves using ATTAs, and to determine whether these proteins were of the predicted size, Western blotting was conducted with total protein as described previously by Guo et al. (2020) (link). For protein separation by SDS-PAGE, 12% separating and 5% stacking gels were used, with proteins transferred to PVDF membranes. Anti-FLAG® M2 primary antibody produced in mouse (Sigma-Aldrich), together with anti-mouse secondary antibody produced in chicken (Santa Cruz Biotechnology, Dallas, TX, United States) and SuperSignal® West Dura Extended Duration substrate (Thermo Fisher Scientific, Waltham, MA, United States), were used for protein detection. Proteins were visualized using an Azure Biosystems c600 Bioanalytical Imaging system (Azure Biosystems, Dublin, CA, United States).