Rabbit anti c1ql2

Briefly, hippocampi from freshly removed brains were dissected in ice-cold PBS, collected in Lysis Buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% triton-X100, 0.1% SDS) and manually homogenated. Samples were centrifuged for 25 min at 13,200 rpm at 4 °C and the supernatant was collected. Protein concentration was calculated with Bradford assay. Protein suspension containing 40 μg of protein was mixed 1:1 with 2 x SDS loading dye (62.5 mM Tris, 10% Glycerol, 5% β-mercaptoethanol, 80 mM SDS, 1.5 mM bromophenol blue), boiled at 95 °C for 5 min, separated by SDS-PAGE and electrophoretically transferred onto PVDF membranes (Merck). Membranes were blocked with 5% non-fat milk (Sigma-Aldrich), incubated with mouse anti-β-actin (1:5000; Sigma-Aldrich) and rabbit anti-C1ql2 (1:500; Sigma-Aldrich), followed by Peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) and developed with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Signal was detected with ChemiDoc Imaging System (Bio-Rad) and analyzed with Image Lab Software (BioRad). Protein signal was normalized with the signal of β-actin.

HEK293 cells were transfected using Lipofectamine 3000 according to the manufacturer’s instructions and were incubated for at least 48 hr. Cells were harvested and proteins were extracted in lysis buffer containing 25 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl₂, 1% Igepal, 5% Glycerol, 1 x EDTA-free proteinase inhibitor, 0.5 mM DTT and 2.5 U/mL Benzonase. Protein A magnetic beads were washed 2 x with PBS including 0.02% Tween-20 and were incubated on a rotating wheel in RT for 2 hr with 2 μg of the following antibodies suspended in 200 μL 2 x with PBS/0.02% Tween-100: rabbit anti-IgG (Cell Signal), rabbit anti-flag (Sigma-Aldrich) or rabbit anti-C1ql2 (Sigma-Aldrich). Beads were washed 2 x with PBS/0.02% Tween-20, resuspended in 50 μL 2 x with PBS/0.02% Tween-20 and 40 μg protein extract was added. Beads were incubated o/n on a rotating wheel at 4 °C. Beads were thoroughly washed with 2 x with PBS/0.02% Tween-20, resuspended in 2 x SDS loading dye, and boiled for 10 min at 95 °C. Western blot (see above) was performed. Briefly, membranes were blocked with 5% non-fat milk (Sigma-Aldrich) and incubated with mouse anti-flag M2 (1:2000; Sigma-Aldrich). Three independent experiments were carried out.