Basic glial cells nucleofector kit

Downregulation of Tmem59 was carried out by electroporation of gene-specific siRNAs using an Amaxa Nucleofector and Amaxa_Cell Line Nucleofector_Kit T (for BV2 cells) or Basic Glial Cells Nucleofector™ Kit (Lonza, for primary microglia), following the manufacturer’s instructions. Each electroporation reaction contained 4 × 10⁶ cells and 200 nM siRNA. The Tmem59 siRNA used was: 5′-GGACCAAGCUGGAAUGUGATT-3′. A scramble siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) was used as control. At 48–72 h after electroporation, cells were subjected to further experiments.

Phospho-CSF1R (Tyr723) (49C10) Rabbit mAb (RRID: AB_2085229), β-catenin (6B3) Rabbit mAb (RRID: AB_331149), anti-Phospho-Akt (Ser473) (RRID: AB_329825) and anti-total-Akt antibodies (RRID: AB_329827) were all bought from Cell Signaling Technology. Anti-GAPDH (RRID: AB_2630358), anti-CSF1R (RRID: AB_2085251), anti-Aβ (MOAB2), anti-β-actin antibodies (RRID: AB_306371) and Alexa Fluor 647 Donkey Anti-Rat IgG H&L (RRID: AB_2813835), as well as the BrdU Cell proliferation ELISA Kit, were all from Abcam. Rat Anti-mouse CD68 monoclonal antibody (RRID: AB_322219) was from Bio-Rad. Anti-HA-tag (RRID: AB_11042321) antibody and anti-Myc-tag antibody (RRID: AB_11182162) were from Proteintech. Rabbit anti-Iba1 (RRID: AB_2687911) was from Wako. Alexa Fluor 488 Goat anti-Rabbit (RRID: AB_10374301), Alexa Fluor 594 Goat anti-Rabbit (RRID: AB_10374440), Alexa Fluor 647 Goat anti-Rabbit antibodies (RRIDAB_10371940) and TRIzol reagent were all from Thermo Fisher Scientific. Granulocyte-macrophage colony stimulating factor (GM-CSF) and CSF1 were from R&D Systems. Basic Glial Cells Nucleofector Kit was purchased from LONZA. ReverTra Ace qPCR RT Master Mix was from TOYOBO. FastStart Universal SYBR Green Master mix was from Roche. CellTiter 96^R Aqueous One Solution (MTS assay) and DeadEnd™ Fluorometric TUNNEL System were from Promega.