- Primary mouse microglia are grown in DMEM (Invitrogen), supplemented with 10% foetal bovine serum (Gibco), 10% horse serum and penicillin and streptomycin (Gibco), at 37°C and 5% CO2 in a 12-well dish.
- Transfect cells with 0.05nmol mouse MAPK11 Silencer Select siRNA (ThermoFisher Scientific, Sample ID ASO10943, ASO1098J and ASO109CX, lot AMO04BXD) using Lipofectamine 3000 (Invitrogen) following the manufacturer´s instructions.
- Lyse cells in 25µl per well cold lysis buffer (10mM Tris-HCl pH 8.0, 150mM NaCl, 1% Nonidet-P40, 1mM EDTA, 10mM NaF, 1mM Na3VO4, protease inhibitors (Sigma).
- Determine total protein content of the lysates using Bradford assay (Bio-Rad, 500-0006, lot 111832).
- Denature 5µg total protein for 5min at 95°C in 5µl Laemmli SDS sample buffer and subsequently separate them on a denaturing freshly cast 10% SDS-PAGE for 1h at 140V.
- Transfer proteins onto PVDF membrane (Pall Life Sciences, 75696G, lot TO3225) with a semi-dry Western blotting system for 50min at 60mA.
- Block the membrane with TBST (TBS, 0.1% Tween) containing 5% milk ON at 4°C.
- Incubation with primary rabbit anti-MAPK11 antibody (antibodies-online, ABIN2798348, lot RAY8110713) diluted 1:500 in TBST containing 5% milk for 1h at RT.
- Wash membrane 5x 5min with TBST.
- Incubation with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (DakoCytomation, P0448, lot 00055814) diluted 1:2000 in TBST containing 5% milk for 1h at RT.
- Wash membrane 5x 5min with TBST and 3x 5min with TBS.
- Reveal protein bands using ECL Prime Western Blotting Detection Reagent (GE Healthcare, RPN2232, lot 9547481) with Curix RP2 Plus medical X-ray films (AGFA Health Care, ENKMV, lot 79040074) on an AGFA Health Care Curix60-developer.
- Strip membranes RestoreTM Western Blot Stripping Buffer (Thermo Scientific, 21059, lot NA 164269) and subsequently incubate with a GAPDH loading control antibody (Sigma, G9545, lot 049K4787) diluted 1:10000 and with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (DakoCytomation, P0448, lot 00055814) diluted 1:2000.
- Wash and reveal the protein bands using self-made ECL Western Blotting Detection Reagent with Curix RP2 Plus medical X-ray films (AGFA Health Care, ENKMV, lot 79040074) on an AGFA Health Care Curix60-developer.