Lsm880 confocal microscope system

The cells grown on collagen-coated Lab-Tek II chamber slides were transfected with plasmids expressing EGFP-tagged proteins for 24–36 h as indicated in figures, The cells were then washed with PBS, fixed with 4% paraformaldehyde in PBS for 15 min. The slides were dehydrated, air-dried, and embedded in DAPI-containing antifadent mounting medium. Images were collected using a Zeiss LSM880 confocal microscope system. The percentage of cells harboring puncta in fluorescence positive cells per image field (40 ×), and the ratio of puncta-like fluorescence intensity to background per cell were quantified by Image J.

FRAP experiments were performed using a Zeiss LSM880 confocal microscope system. Spots of approximately 2 μm diameter in droplets of approximately 10 μm were photobleached with 20% laser power for 1 s using 488 nm lasers. Time-lapse images were acquired over 1 min after bleaching. Images were processed using the ImageJ, and the FRAP data were fitted to a single exponential model using the GraphPad Prism 9.

Purified proteins were diluted to indicated concentrations in buffer (25 mM Tris-HCl, pH 5.5, 150 mM NaCl, 1 mM DTT, unless specified otherwise) at room temperature. The protein solution (5 μl) was loaded onto a glass slide, covered with a coverslip, and imaged using a Zeiss LSM880 confocal microscope system.
Phase separation of recombinant SARS2-NP with Cy5-labeled SARS-CoV-2 RNA (5′-CACUCGCUAUGUCGAUAACAACUUCUGUGGCCCUGAUGGCUACCCUCUUGAGUGCAUUAAAGA-3′) was performed in the foregoing buffer. The mixture was transferred to a 96-well plate and imaged. The occupied area, or equivalent diameter (EqDiameter, the diameter of a circle with the same area as the measured object) of droplets per image (40 ×) were processed by Image J with particle analysis tool.

The cells were grown on collagen-coated Lab-Tek II chamber slides (Nunc, 154453), washed twice with PBS, and fixed in 4% formaldehyde in PBS for 15 min at room temperature. Subsequently, the slides were washed with TBS (25 mM Tris, pH 7.4, 100 mM NaCl), and permeabilized for 15 min in TBS containing 0.1% Triton X-100, and washed with TBST (0.05% Tween 20 in TBS). The slides were then blocked for 2 h with 0.5% milk powder in TBST and subsequently incubated with the appropriate combinations of antibodies overnight at 4 °C. After washing with TBST, proximity ligation was performed using Rabbit PLUS and Mouse MINUS Duolink in situ PLA kits (Sigma, DUO94102), according to the manufacturer’s instructions. Subsequently, the slides were dehydrated, air-dried, and embedded in DAPI-containing antifadent mounting medium (VectorLabs, H-1200). PLA signals were imaged by a Zeiss LSM880 confocal microscope system. The PLA-detected proximity (PROX) complexes (red fluorescent dots) intensity per image (40 ×) was quantified by Image J with particle analysis tool.

Immunostaining was carried out as described in our previous work (36 (link)). Briefly, striatal cells were grown on poly-d-lysine (0.1 mg/ml)–coated glass coverslips, and after 48 h of transfection, the medium was changed to Krebs buffer medium devoid of serum and AAs for 1 h to induce full starvation conditions. For the stimulation conditions, cells were stimulated with 3 mM l-leucine for 15 min. Cells were washed with cold PBS, fixed with 4% paraformaldehyde (20 min), treated with 0.1 M glycine, and permeabilized with 0.1% (v/v) Triton X-100 (5 min). After being incubated with blocking buffer (1% normal donkey serum, 1% [w/v] bovine serum albumin, and 0.1% [v/v] Tween-20 in PBS) for 1 h at room temperature, cells were stained overnight at 4 °C with antibodies against HA (CST, 3724; 1:1000 dilution), pS6^Ser235/236 (CST, 4858S; 1:200 dilution), pAKT^Ser473 (CST, 4060S; 1:500 dilution), and mTOR (CST, 2983S; 1:200 dilution). Alexa Fluor 488 (Invitrogen, A21202; 1:1000 dilution) or Alexa Fluor 568 (Invitrogen, A10037; 1:1000 dilution)–conjugated secondary antibodies were incubated together with the nuclear stain 4′,6-diamidino-2-phenylindole for 1 h at room temperature. Glass coverslips were mounted with Fluoromount-G mounting medium (ThermoFisher Scientific, catalog no.: 0100-01). Images were acquired by using the Zeiss LSM 880 confocal microscope system with 63× objective.