N1E cells for detection of recombinant LPPR3
DIV 0: N1E cells were plated at a density of 150 000 cells/well (6-well plates) and grown overnight in DMEM medium (Gibco) with 10% FCS and 1% penicillin/streptomycin.
DIV 1: The cells were transfected with 1 µg of DNA using Lipofectamine 2000 (ThermoFisher Scientific) according to manufacturer´s protocol. The cells were grown overnight.
DIV 2: The cells were harvested.
Primary hippocampal neurons for detection of endogenous LPPR3
DIV 0: Cells were plated at a density of 500 000/well (6-well plates) and grown in Neurobasal A medium (Gibco) supplemented with 2% B27, 1% Glutamax and 1% penicillin/streptomycin for 9 days.
DIV 9: The cells were harvested.
Ripa buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% sodium dodecyl sulphate (SDS))
phosphatase inhibitors (1 mM Na2MO4,1 mM NaF, 20 mM β-glycerophosphate, 1 mM Na3VO4, 500 nM cantharidin)
protease inhibitors (Calbiochem set III, dilution 1:100)
All steps were carried out on ice. Cell culture medium was aspirated and the cells were quickly washed with ice cold PBS. 200 µl (neurons) or 300 µl (N1E cells) lysis buffer was added to each well, the cells were scraped off and collected. The homogenate was rotated at 4 degrees for 20 minutes and then centrifuged at 14 000 rpm at 4 degrees for 20 minutes. The supernatant was collected into a fresh Eppendorf tube.
Protein quantification was carried out using the BCA Thermo Scientific Pierce™ Protein Assay according to manufacturers protocol.
50 µl of sample was mixed with 50 µl 4x Roti Load sample buffer and boiled at 95 degrees for 5 minutes.