A full-length rat Pdlim2 coding sequence was cloned into a bacterial expression vector (pCAL-n-FLAG; Stratagene, La Jolla, CA). Recombinant protein fused to the 5-kDa tag, which included the calmodulin-binding peptide and FLAG peptide, was purified from induced cultures of E. coli cells (Rosetta-gami B(DE3), Novagen, San Diego, CA) using calmodulin affinity resin (Stratagene). Corneas from five adult Wistar rats or approximately 100 mg of rat lung were homogenized in 1 mL of a solution containing 50 mM Tris (pH 8.0), 300 mM NaCl, 5 mM EDTA, 0.4% Triton X-100, 0.4% Tween 20, and a protease inhibitor cocktail tablet (Roche, Indianapolis, IN). Extracts were centrifuged at 15,000g for 20 minutes at 4°C. Five μg of purified Pdlim2 or FLAG-Bacterial alkaline phosphatase fusion protein (Sigma, St. Louis, MO) were added to 0.5 mL of supernatants and incubated for 1 hour at 4°C with constant mixing. Then 50 μL of the anti-FLAG agarose beads (Sigma) were added, and incubation continued overnight in the same conditions. Beads were washed five times with the buffer used for binding. Beads were boiled in 20 μL 2x Laemmli sample buffer (Invitrogen) for 5 minutes and centrifuged briefly. Ten μL of the supernatant were loaded on 10% SDS-polyacrylamide gel. Gels were stained with Colloidal Blue (Invitrogen) and the most prominent bands were cut from the gels. Protein identification was performed as a service by ProtTech, Inc. (Norristown, PA) using the nanoflow liquid chromatography and tandem mass spectrometry (LC-MS/MS) technique (see http://www.prottech.com for details).
Full-length rat α-actinin-1 and α-actinin-4 cDNAs (gw08h06 and gw13b12, respectively) were identified in the rat eye angle library. Full-length cDNA for α-actinin-2 was amplified from rat embryo random primed cDNA. These three cDNAs were cloned into the pcDNA3.1/Myc-His(+)B vector (Invitrogen). [35S]-labeled full-length α-actinins were synthesized using a rabbit reticulocyte lysate (TnT Rabbit Reticulocyte Lysate System; Promega, Madison, WI). Labeled proteins were purified using G-25 spin columns (Amersham Biosciences, Piscataway, NJ). [35S]-labeled actinins were incubated with approximately 5 μg of the purified Pdlim2 protein for 1 hour at 4°C in 0.5 mL of the binding buffer containing 50 mM Tris (pH 8.0), 200 mM NaCl, and 0.4% Triton X-100 with constant mixing. Then 50 μL of the anti-FLAG beads were added to each tube and incubation continued overnight at 4°C. The agarose beads were washed five times in the binding buffer. Proteins were eluted by boiling in the SDS-PAGE loading buffer and loaded onto SDS-PAGE gels. Gels were dried after electrophoresis and radioactive proteins were visualized by autoradiography. Efficiency of GST-pull down was estimated by comparison with a 10% input lane.