Anti ha high affinity antibody clone 3 f10

Cells were lysed with Non-ident NP40 buffer as previously described [25 (link)]. Xenograft tissues were lysed similarly, including a previous step of mechanical disruption. Total protein concentrations were determined with the Bio-Rad protein quantification reagent (Bio-Rad Laboratories, Inc.). Equal amounts of protein (30–50 μg) were resolved by SDS-PAGE on 7–12% gels under reducing conditions. Separated proteins were transferred onto PVDF membranes, blocked with 5% milk or BSA in TBS/T, and incubated overnight with the following primary antibodies: p-Y1234/Y1235 MET, p-Ser473 AKT, p-Thr202/Tyr204 ERK1/2, p-Ser235/236 S6, pan-AKT, and pan ERK (all from Cell Signaling Technology). Anti-β-actin antibody was obtained from Millipore Corporation. The anti-HA high affinity antibody (clone 3 F10) was purchased from Roche.
Membranes were incubated with appropriate secondary antibodies and signals were detected with the ECL kit (Amersham Pharmacia Biotech) or with infrared fluorescence on an Odyssey imager (Li-Cor biosciences).
Immunoprecipitation was performed using the Dynabeads® Protein G kit following manufacturer’s instructions (ThermoFisher Scientific, #10003D).
With the exception of samples from in vivo tumors and organotypic slices, immunoblots shown are representative of at least three independent experiments.

The DBA/2 mice were injected into the ophthalmic venous sinus with 10⁷ plaque-forming units of rAd5-CMV-PLAP-aMh-FcG2a or rAd5-CMV-PLAP-aMh-ILZ-HA previously dialyzed against PBS. As a control, we used mice injected with PBS. At 2 days (48 h), 7 and 14 days after the inoculation, serum samples and vaginal washes were collected, separated from cell debris by centrifugation, and serial sample dilutions were used for titration on ELISA with LAMPs or bovine serum albumin coated at a concentration of 100 ng/well. As a secondary antibody, we used polyclonal goat anti-mouse IgG (Fc specific) antibody horseradish peroxidase conjugate (Sigma-Aldrich, St. Louis, MO, USA) or anti-HA high affinity antibody (clone 3F10) (Roche, Indianapolis, IN, USA). The rAds injection and sample collection protocol was approved by the Gamaleya Research Center of Epidemiology and Microbiology Institutional Animal Care and Use Committee (IACUC) and was performed under Protocol # Imb-2014-048.