F3040

Cells were lysed with Laemmli sample buffer. Protein concentrations in the cell lysates were measured with the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples were boiled for 5 min in sample buffer containing bromophenol blue and 1 × β-ME, and equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretic separation was carried out on 10% polyacrylamide gel (Bio-Rad Laboratories), and the proteins were subsequently transferred to Immobilon-P membrane (Millipore, Billerica, MA, USA). Membranes were blocked in phosphate-buffered saline (PBS) with Tween 20 (PBST) with 5% nonfat dry milk powder and incubated with the following primary antibodies: HMGA2 (1:250 dilution; sc-30223, Santa Cruz, Dallas, TX, USA) or FLAG (1:1000 dilution; F3040, Sigma-Aldrich, St. Louis, MO, USA). The primary and secondary antibodies for HMGA2 were diluted with Can Get Signal (Toyobo, Osaka, Japan). The membranes were then washed with PBST and incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibody (GE Healthcare, Little Chalfont, UK). Antibody binding was detected with the enhanced chemiluminescence detection system (ECL and ECL Plus; GE Healthcare).

The following antibodies were used: anti-FUS N-terminal [western blot (WB) 1:5000; catalog no. NB100-565, Novus Biologicals], anti-FUS C-terminal [immunofluorescence (IF) 1:300, WB 1:5000; catalog no. NB100-562, Novus Biologicals], anti-FUS (IF 1:400; catalog no. sc-47711, Santa Cruz Biotechnology), anti-∆14 [IF 1:300 (13 (link))], β3-tubulin [1:1000; catalog no. 801202 (BioLegend); 1:500; catalog no. 302 306 (SySy); and 1:2000; catalog no. 119-154886 (Raybiotech)] anti-GFP (IF 1:1000; catalog no. GFP1011, Aves Labs), anti-FMRP (IF 1:300 and WB 1:1000; catalog no. ab17722, Abcam), anti-SMN1 (IF 1:300 and WB 1:1000; catalog no. 610646, BD Biosciences), anti-Flag M1 (IF 1:500; catalog no. F3040, Sigma), anti-G3BP1 (1:200; catalog no. 611126, BD Biosciences), anti-RPL26 (IF 1:800 and WB 1:2000; catalog no. ab59567, Abcam), anti-RPS6 (WB 1:1000; Cell Signaling Technology), anti-LAMP1 (IF 1:300; catalog no. ab25245, Abcam), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (WB 1:5000; mab374, Millipore), anti-HA [WB 1:3000 and immunohistochemistry (IHC) 1:100; catalog no. H6908, Sigma-Aldrich], and anti-ChAT (IHC 1:100; Chemicon). Alexa Fluor–conjugated secondary antibodies were from Invitrogen (1:1000) or Jackson ImmunoResearch (1:500).

Hippocampal neurons transfected with TrkB-Flag were kept in Neurobasal media for 1 hr and then incubated on ice with 1:50 mouse anti-Flag antibody (Sigma-Aldrich, Cat# F3040, RRID:AB_439712). In selected samples, 20 nM H_CT was also added (Deinhardt et al., 2006 (link)). Internalisation of receptors was then induced by incubation with 50 ng/mL BDNF for 30 min at 37°C. Antibodies bound to receptors still at the cell surface were dissociated by washing twice for 1 min in PBS supplemented with 1 mM EDTA. In experiments to measure recycling of internalised receptors, neurons were further stimulated with BDNF for other 60 min in the presence of an Alexa Fluor647-conjugated anti-mouse secondary antibody, fixed, permeabilised, and incubated with an Alexa Fluor555-conjugated anti-mouse secondary antibody to detect total internalised receptor.

Omalizumab, ligelizumab, and quilizumab were prepared as IgG1-format antibodies, as described above. Edox cells were harvested and incubated with antibodies at the concentration corresponding to saturation concentration for staining (10 nM for 15cl12, 3.33 nM for omalizumab, 1.5 nM for ligelizumab, and 100 nM for quilizumab), in RPMI with 10% FCS and 2 mM L-glutamine, sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 200,000 cells/well, on ice, and at 37 °C for 4 h. Samples were then placed on ice for 5 min, resuspended in 10 µg/mL anti-FLAG antibody (RRID: AB_439712, F-3040, Sigma-Aldrich) in 2% BSA–PBS, and incubated on ice for 30 min. The binding of anti-FLAG was detected with goat anti-mouse-FITC conjugate (AB_259490, F-2653, Sigma-Aldrich), diluted 1:200 in 2% BSA–PBS, after 30 min incubation on ice. Cells were resuspended in 200 µL ice-cold PBS and analyzed with a Guava^® easyCyte™ Flow Cytometer (Luminex). A decrease in fluorescent signal upon treatment at 37 °C was calculated as (1-(MFI at 37 °C/MFI on ice)) × 100.

To confirm the formation of the intramolecular adduct in Opn5L1, we prepared Opn5L1NC and C188T mutant constructs that contained a factor Xa recognition site and a FLAG tag sequence. Hereafter we refer to the protein expressed from the Opn5L1NC construct as Opn5L1_XaFLAGicl3. Experimental procedures for factor Xa cleavage and western blotting were as follows. The purified Opn5L1_XaFLAGicl3 (20 μL) was irradiated with light passed through a glass cut-off filter VY52 at room temperature for 1 h in the presence or absence of 8 mM 2-picoline borane. The samples were then incubated at 37 °C for 2 h in the presence or absence of factor Xa (final concentration: 50 μg mL^-1). The samples after incubation were mixed with SDS sample buffer containing DTT (final concentration: 200 mM). Finally, aliquots of the samples were subjected to SDS-PAGE. The protein fragments separated by SDS-PAGE were transferred to a polyvinylidene difluoride membrane, and analyzed by standard western blotting procedures using anti-FLAG M1 (10 μg mL⁻¹; Sigma F3040) and Rho1D4 (10 μg ml⁻¹)^{50 (link)} antibodies.