After washing in TBS-T and probing with the corresponding horseradish peroxidase-labeled secondary antibody (Pierce Biotechnology, Rockford, IL), bound antibodies were identified using Amersham ACL Plus Western Blotting Detection Reagents (GE Healthcare, Pittsburgh, PA) and luminography.
Lymphocytes were stimulated with either plate bound anti-CD3 (OKT3 – Insight Biotechnology) and soluble anti-CD28 (BD Pharmingen) or anti-CD3/CD28 activation beads (Invitrogen).
Antibodies (Sc73 and Sc-372 from Santa-Cruz Biotechnology for JunB and p65/RelA, respectively) were previously bound to Dynabeads (Invitrogen) according to the supplier's recommendations.
For FACS analysis, cells were incubated with anti-PECAM (Santa Cruz biotechnology), anti-VE-cadherin (Santa Cruz Biotechnology) or anti-SMA (Abcam) and bound secondary anti-mouse Alexa 660 or anti-rabbit Alexa 555 (Molecular Probes).
Equal amounts of protein from the stria vascularis capillary lysates prepared with RIPA buffer were incubated on a rotator overnight with an antibody bound to protein A agarose beads (Cat# sc-2001, Santa Cruz Biotechnology, Inc) at 4°C.
Total TNF-α (free TNF-α and TNF-α bound to sTNF-R) was determined using a commercially available ELISA (HyCult Biotechnology BV, Uden, The Netherlands).
Immunoprecipitation of RNA bound to endogenous AUF1 immunoprecipitation was performed by cross-linking anti-AUF1 antibody (Upstate Biotechnology) to protein G-agarose beads using dimethylpimelimidate (Sigma-Aldrich).
Membranes were then washed three times at room temperature in TBST for 30 min each time, and bound Ig was detected using anti-isotype monoclonal secondary antibody coupled to horseradish peroxidase (Santa Cruz Biotechnology; Heidelberg, Germany).
Cells were washed with PBS to remove unbound antibody, and bound rabbit immunoglobulin G (IgG) was detected with goat anti-rabbit IgG-fluorscein isothicyanate (FITC) conjugate (Santa Cruz Biotechnology Inc, Santa Cruz, USA) used at 1∶400 dilution.
Membrane bound HRP-conjugated secondary antibody was detected using SuperSignal West Femto Maximum Sensitivity Substrate (cat. #34095, Pierce Biotechnology, Inc., Rockford, IL).
The amount of PTEN protein that bound to the LMVs was then visualized by western blot using an anti-PTEN antibody from Santa Cruz Biotechnologies SC-68188).
