Egta e3889

Mme BMDMs were washed with PBS, dissociated by lysis buffer (50 mM Tris (T5941) pH 6.8, 2% SDS (L3771), 5% glycerol (G5516), 2 mM DTT (D0632), 2.5 mM EDTA, 2.5 mM EGTA (E3889), all reagents from Sigma-Aldrich), supplemented protease and phosphatase inhibitor cocktail (Sigma-Aldrich, P8340, 524629). Samples were boiled for 10 min with SDS-sample buffer at 100 °C. The samples were separated by 12% SDS-PAGE and transferred to PVDF membrane (Bio-Rad, 1620177). The membrane was blocked with TBST buffer (10 mM Tris, pH 8.0, 0.15 M NaCl (S7653), and 0.05% Tween 20 (P1379); all reagents from Sigma-Aldrich) containing 5% BSA for 1 h at room temperature and then probed with anti-phospho(Tyr416)-Src, c-Src (Cells Signaling Technology, 2101, 2108) and beta-actin (Sigma-Aldrich, 5441) primary antibodies overnight at 4 °C. beta-actin was used for the loading control. After washing with TBST buffer, blots were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Proteins were detected with chemiluminescence (Immobilon Western Chemiluminescent HRP Substrate, Merck Millipore, WBKLS0500). The pixel density of bands was determined by Image J software.

A list of oligonucleotides and antibodies used for the study are listed in Supplementary Tables 2, 3, respectively. K252a (1863), thapsigargin (1138), U73122 (1268/10) and U73343 (4133/10) were from Tocris. Fluo-4-AM (F14201), Powerload (P10020) and Probenecid (P36400) were from Thermo Fisher Scientific. EGTA (E3889) was from Sigma-Aldrich. Brain-derived neurotrophic factor (BDNF) (B-250) was from Alomone labs.

Cells (2 × 10³) were grown in 100 mm dishes. Following metformin treatment, cells were harvested and centrifuged at 700× g at 4 °C for 5 min. Pellet was resuspended on ice in 200 μL of a solution containing 2 mM magnesium chloride (MgCl₂, M8266; Sigma-Aldrich), 10 mM potassium chloride (KCl, P9333; Sigma-Aldrich) and 10 mM Tris pH 7.4. After 10 min, each samples were mixed with 200 μL of a solution containing 400 mM sucrose (S5390; Sigma-Aldrich), 10 mM Tris pH 7.4, 2 mM EDTA, 2 mM ethylene glycol-bis(2-aminoethylether)-N, N, N’,N’-tetraacetic acid (EGTA, E3889; Sigma-Aldrich), 2 mM PMSF, 20 mM NaF, 2 mM Na₃VO₄ and PI. Cells were broken with 50 Dounce strokes on ice. Homogenates were transferred into 1.5 mL tubes and centrifuged at 900× g for 10 min at 4 °C. Pellet were discarded and supernatant fractions were transferred in new 1.5 mL tubes and centrifuged at 17,000× g for 30 min at 4 °C. Pellet (mitochondrial fractions) were lysed in 20 μL of lysis buffer and protein concentration determined by the Bradford assay.