Sodium orthovanadate sov

Studies used previously generated Chinese hamster ovary cells stably expressing human IR (CHO IR cells) (52 (link)). 293T cells stably expressing human IR (293T IR cells) with a C terminal streptavidin-binding peptide (SBP) tag were generated previously (25 (link), 53 (link)). bSM, eSM, POPC, DOPC, DLPC, DMPC, DPPC, DSPC, and cholesterol were from Avanti Polar Lipids. MαCD was purchased from AraChem. Methotrexate and sodium orthovanadate (SOV) were purchased from Sigma Aldrich. Dulbecco’s modified eagle medium (DMEM, 4.5 g/l glucose, L-glutamine, sodium pyruvate), phosphate buffered saline (PBS) without calcium and magnesium (0.144 g/l KH₂PO₄, 9 g/l NaCl, 0.795 g/l Na₂HPO₄ (anhydrous)), trypsin-EDTA, antibiotic-antimycotic solution, and L-glutamine were purchased from Corning. Nonessential amino acids and ham’s F12 media were purchased from Gibco. G418 (geneticin) was from Goldbio, fetal bovine serum (FBS) was from VWR international, TDM and HDM were from Anatrace. DM was from Dojindo Molecular Technologies, DDM was from Thermo Fisher.

Recombinant human TGF-β1 was obtained from R&D systems (Minneapolis, MN, USA). Bleomycin sulfate was obtained from Hisun Pharm (Taizhou, China). Sodium orthovanadate (SOV) was obtained from Sigma-Aldrich (Shanghai, China). Wiskostatin (Wis) was obtained from Abcam (Shanghai, China). N-WASP was obtained from Proteintech (Wuhan, China). Goat anti-rabbit IgG of β-actin, Alpha-smooth muscle actin (α-SMA), p^Y256N-WASP and GAPDH were purchased from Affinity Biosciences (Changzhou, China). Goat anti-mouse IgG of Vimentin and E-cadherin were purchased from Cell Signaling Technology (Shanghai, China), Goat anti-rabbit IgG of N-cadherin, Fibronectin and Collagen I were purchased from Cell Signaling Technology (Shanghai, China), TUFT1 was purchased from Thermo Fisher (Suzhou, China).

Schaffer collateral fibers in hippocampal slices were electrically stimulated and evoked field excitatory postsynaptic potentials (fEPSP) were recorded in the stratum radiatum of hippocampal CA1. Two glass micro-electrodes (tip resistance 5-6 MΩ) for stimulation and recording were filled with ACSF and carefully positioned in the slice and placed at depths where imaging was performed. The current pulses (5–15 pulses, 0.2-0.3 ms in duration) were delivered via the stimulating electrode from a stimulus isolator (AMPI; Science Products). The stimulus strength varied between 10–40 µA. Field potentials were recorded using a patch clamp amplifier (Multiclamp 700B; Molecular Devices).
To block calcium extrusion from cells, we applied two calcium-pump blockers: 5 µM sodium-orthovanadate (SOV, Sigma Aldrich) and 50 µM benzamil hydrochloride hydrate (BHH, Sigma Aldrich) dissolved in HEPES-based ACSF. To block voltage-gated sodium channels, we applied 1 µM tetrodotoxin (TTX, Hello Bio). Inhibition of voltage-gated calcium channels was done using 100 µM CdCl₂ (Sigma Aldrich). Finally, 20 µM cyanquixaline (CNQX, Sigma Aldrich) was used to block AMPA receptors.