Secondary anti mouse antibody

Protein–lipid overlay assays with PIP and lipid strips (P-6001 and P-6002 respectively, Echelon Biosciences) were performed according to the manufacturer’s instructions. Briefly, strips were first blocked with 3% fatty acid-free BSA in PBS (3 ml, 10 mM phosphate, and 150 mM NaCl, pH 7.4) for 1 h and incubated 2 h at room temperature with blocking buffer containing 0.5 μg/ml for each of GST-EXO70B1-ct, GST-EXO70B2-ct, HA-EXO70B1, and HA-EXO70B2. The strips were washed 3× with 3 ml of PBS with 0.1% Tween. To detect the proteins, an anti-GST (Echelon Biosciences, dilution 1/2000) or anti-HA mouse monoclonal antibody (Thermo Fisher Scientific, dilution 1/1,000 dilution) was used. Subsequently, chemiluminescence detection (ECL, Amersham) of the secondary anti-mouse antibody (Promega) conjugated with horse radish peroxidase was used for identification of positive interactions. The signal was documented using Bio-Rad documentation system.

About 5 × 10^6 cells were collected and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.8, 50 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.5% NP-40) and Western blotting analysis was performed as described previously (Pratt et al., 2012 (link)). The blots were detected with mouse monoclonal anti-IPO7 (Sigma) directed to the C-terminus at 1:1,000 dilution, or mouse monoclonal anti alpha-tubulin (Sigma) at 1:10,000 dilution, followed by a secondary anti-mouse antibody (Promega) at 1:5,000 dilution.

Twenty-microgram samples of total proteins, prepared as described above, were loaded on 10% polyacrylamide-SDS denaturing gels. After electrophoresis, samples were blotted onto polyvinylidene difluoride (PVDF) membranes (0.45 µm pore-size, Amersham Biosciences, Little Chalfont, United Kingdom). Transfer of proteins to the membrane and concentrations of the samples were visualized by staining the membranes with Ponceau red for 10 min. Finally, membranes were immunostained with a mouse monoclonal anti-MMP3 antibody (anti-MMP-3 (Ab-1) Mouse mAb (55-2A4) IM36 from Calbiochem, CA, USA). The secondary anti-mouse antibody was from Promega (St. Louis, MO, USA).