Anti n wasp

HeLa cells were maintained in minimal essential medium (MEM) and MEFs, and A549 cells were maintained in Dulbecco’s Modified Eagle Medium. All cells were supplemented with 10% FBS (fetal bovine serum), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C and 5% CO₂. The HeLa cell line stably expressing LifeAct-iRFP670 (37 (link)) was previously generated in the Way lab. Nck −/− MEFs (67 (link)) and N-WASP−/−MEFs (68 (link)) were provided by the late Tony Pawson (Samuel Lunenfeld Research Institute, Toronto, Canada) and Scott Snapper (Harvard Medical School, Boston, MA, USA), respectively. A549 cells were obtained from ATCC. For this study, lentiviral expression vectors were used to stably express TagGFP2-Nck in Nck−/− MEFs and TagGFP2-N-WASP in N-WASP−/− MEFs. All cell lines were generated using the lentivirus Trono group second generation packaging system (Addgene) and selected using puromycin resistance (2 µg/mL) as previously described (69 (link)). Expression of the relevant fusion proteins was confirmed by live imaging and immunoblot analysis (Fig. S4). The following primary antibodies were used: anti-Nck (BD transduction; 1:1,000), anti-vinculin (Sigma #V4505; 1:2,000), anti-N-WASP (Cell Signalling #4848S; 1:1,000), and anti-TagGFP2 antibody (Evrogen #AB121; 1:3,000). HRP-conjugated secondary antibodies were purchased from The Jackson Laboratory.

At 8 (HeLa) or 16 hours (MEFs) post-infection, cells were fixed with 4% paraformaldehyde in PBS for 10 minutes, blocked in cytoskeletal buffer (1 mM MES, 15 mM NaCl, 0.5 mM EGTA, 0.5 mM MgCl₂, and 0.5 mM glucose, pH 6.1) containing 2% (vol/vol) fetal calf serum and 1% (wt/vol) BSA (bovine serum albumin) for 30 minutes, and then permeabilized with 0.1% Triton-X/PBS for 5 minutes. To visualize CEV, cells were stained with a monoclonal antibody against B5 [19C2, rat, 1:1,000 (5 (link))] followed by an Alexa Fluor 647 anti-rat secondary antibody (Invitrogen; 1:1,000 in blocking buffer) prior to permeabilization of the cells with detergent. Other primary antibodies used were anti-Nck (Millipore #06-288; 1:100) and anti-N-WASP (Cell Signalling #4848S; 1:100) followed by Alexa Fluor 568 conjugated secondary antibodies (Invitrogen; 1:1,000 in blocking buffer). Actin tails were labeled with Alexa Fluor 488 phalloidin (Invitrogen; 1:500). Coverslips were mounted on glass slides using Mowiol (Sigma). Coverslips were imaged on a Zeiss Axioplan2 microscope equipped with a 63×/1.4 NA Plan-Achromat objective and a Photometrics Cool Snap HQ cooled charge-coupled device camera. The microscope was controlled with MetaMorph 7.8.13.0 software.