Click it plus opp alexa fluor 594 protein synthesis assay kit

For microscopic analyses, HeLa cells were plated on coverslips in 24-well plates and transfected with SMPX-V5 constructs (500 ng/well) using FuGENE 6. Three days after transfection, the cells were treated with 20 µM MG132 (Sigma-Aldrich) for 2 h or left untreated. Cells were washed with PBS and fixed with 4% PFA for 15 min. Immunofluorescence stainings were performed with the following antibodies: V5 mouse mAb (RRID:AB_2556564), V5 rabbit pAb (Sigma-Aldrich AB3792, RRID:AB_91591), TIA1 goat pAb (Abcam ab61700, RRID:AB_945832), TIAL1 rabbit pAb (Abcam ab26257, RRID:AB_470826), TIAL1 rabbit mAb D32D3 (RRID:AB_10839263), G3BP mouse mAb (Abcam ab56574, RRID:AB_941699), HNRNPA1 mouse mAb 9H10 (Abcam ab5832, RRID:AB_305145), eIF3η goat pAb (RRID:AB_671941), and Oligomer A11 rabbit pAb (Thermo Fisher AHB0052, RRID:AB_2536236). Nascent proteins were labeled and detected with the Click-iT Plus OPP Alexa Fluor 594 Protein Synthesis Assay Kit (Thermo Fisher) according to the manufacturer’s instructions. Images were acquired with Zeiss Axio Imager M2 using 40× NA 1.30 and 20× NA 0.80 objectives, or with a Leica TCS SP8 confocal microscope using a 40× NA 1.10 objective (Leica Microsystems).

The protein synthesis in fly brains was assessed using the Click-iT Plus OPP Alexa Fluor 594 Protein Synthesis Assay Kit (Thermo Fisher Scientific, C10457). Fly brains were dissected in Drosophila medium (Gibco, 21720024) and then incubated in a medium containing 1:1000 (20 µM) of Click-iT OPP reagent at room temperature for 30 min. The brains were washed three times with PBS, and then fixed with 4% PFA (Biosharp, BL539A) for 1 hr at room temperature. The brains were permeabilized and blocked in 5% BSA (Biofroxx, 4240GR005) in 0.3% PBST (PBS with 0.3% Triton X-100) for 90 min at room temperature, and then washed three times with PBS. The brains were incubated with primary antibodies (GFP, 1:200, Cell Signaling Technology, 2955) overnight at 4℃, washed three times with PBS, and incubated with secondary antibodies (1:200, Thermo Fisher Scientific) and DAPI (1:500, Sigma, D9542) for 1 hr at room temperature. For the Click-iT reaction, brains were incubated in the Click-iT reaction cocktail in the dark at room temperature for 30 min. Brains were then washed three times with PBS and imaged by confocal fluorescence microscopy (ZEISS LSM880).