Dynabeads m 450

The E236K mutation was introduced into the plasmid pIRES-KCNA2-AcGFP1 containing the full-length WT hKv1.2 cDNA using the QuickChange™ site-directed mutagenesis kit (Stratagene Cloning Systems, Santa Clara, CA, USA). The complete coding region of the cDNA was sequenced to exclude polymerase errors. HEK293 cells were transiently transfected with the Kv1.2 WT or E236K (7 μg) and CD8 reporter plasmids (1 μg) using the calcium–phosphate precipitation method. Only cells bound with anti-CD8 antibody-coated microbeads (Dynabeads M450, ThermoFisher Scientific, Waltham, MA, USA) were used for patch-clamp recordings.

All experiments were performed in CHO-K1 (Chinese Hamster Ovary, CHO) cells obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured at 37°C in Iscove’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) L-Glutamine (Gibco), and antibiotics (100 IU/ml penicillin and 100 mg/ml streptomycin; all from Gibco, Paisley, UK) in a 5% CO₂ atmosphere.
Cells were transiently cotransfected with K_V4.3 (cloned into pEGFPn1, gently given by Dr. D.J. Synders, University of Antwerpen, Belgium) and DREAM (cloned into pcDNA3.1). In both cases, cells were cotransfected with EBO-pcDLeu2 as a reporter gene, codifying CD8. Transfection was performed using Fugene-6 (Promega) following manufacturer’s instructions as previously described (Moreno et al., 2017 (link); López-Hurtado et al., 2018 (link)). After 48 h transfection, cells were removed from culture plates using TrypLE™ Express (Life Technologies), after exposing them to polystyrene microspheres bound to anti-CD8 (Dynabeads M-450, Thermo Fisher Scientific; Franqueza et al., 1999 (link); Naranjo et al., 2016 (link)). Because the level of expression of DREAM can be crucial for the effects of IQM-266, only cells cotransfected with K_V4.3 and DREAM that exhibit a recovery kinetics from inactivation between 20 and 45 ms were selected for electrophysiological recording.

Magnetic beads (4.5 µm, Dynabeads M-450, Thermo Fisher Scientific) were coated with fibronectin (Gibco, PHE0023) following the manufacturer’s instructions. Suit2 cells were incubated with fibronectin-coated beads for 30 min at 37 °C and then thoroughly washed with PBS to remove unbound beads. Individual cell-bound beads were then subjected to a pulsatile force regime using magnetic tweezers consisting of a 3 s, 6 nN force pulse, followed by a 4 s rest period, repeated for 12 pulses over ~100 s. The bead trajectories were recorded using an inverted microscope (Nikon Ti-Eclipse, C-LHGFI HG Lamp, CFI Plan Fluor 40× NA 0.6 air objective) fitted with a Neo sCMOS camera (Andor) with NIS elements AR software, and analysed using a custom MATLAB script. The amplitudes of each pulse were extracted from bead trajectories and normalised to the 1st pulse. The amplitudes of the 1st and 12th pulse were compared to quantify the decrease in amplitude of the bead movement as a result of cytoskeletal reinforcement.

Anti-CD3ɛ (OKT3), anti-CD137 (6B4), and mIgG1 isotype control (MOPC-21) mAbs were covalently coupled to Dynabeads M-450 and M-280 Tosylactivated (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Unless otherwise referred, all antibodies used in WB assay were diluted at 1:800. Rabbit polyclonal antibodies recognizing CHML (HPA029628), Rab14 (R0656) were purchased from Sigma-Aldrich. Anti-REP2 for endogenous immunoprecipitation was from Absci (AB41393). Anti-Rab1B (A7514), anti-Rab5A (A1180), anti-Rab5B (A7447), anti-Rab27A (A1934), anti-Rab31 (A7506) were from Abclonal. Anti-Rab4A (10347-1-AP), anti-Rab7A (55469-1-AP), anti-Flag (20543-1-AP) were from Proteintech. Anti-Rab4B (sc-376386), anti-Rab11A (sc-166912), anti-GAPDH (sc-47724), anti-α-tubulin (sc-73242), anti-β-actin (sc-58673) were from Santa Cruz. Anti-GST was from Sangon (D199985). Anti-Flag M2 (A2220) beads was from Sigma Aldrich. Glutathione Sepharose 4B, rProtein A sepharose, protein G were from GE. Dynabeads-M450 was from Thermo Fisher. Amicon ultra-0.5 and Amicon 10K ultra-15 were from Millipore.

Anti-Rab14 (Sigma-Aldrich) antibody or the control Rabbit IgG was ligated to Dynabeads M450 (Thermo Fisher) according to the manufacturer’s protocol. Briefly, 200 µg goat anti-Rabbit antibody was incubated with 1 mL beads in 1 mL 0.1 M borate buffer for 30 min, then adding 20 µL 5% BSA, rotating at room temperature for 48 h. 200 µL beads were continued to incubate with 10 µg Rabbit Anti-Rab14 antibody or control Rabbit IgG at 4 °C overnight. Endomembrane was prepared as described previously with some modifications⁶⁰ . Briefly, 3 × 10⁸ PLC/PRF/5 cells were washed by pre-cold PBS, scrapped in 1 mL cold PBS, then incubated with hypotonic buffer (10 mM Tris–HCl) for 2 min, the pelleted cells were then resuspended in homogenesis buffer (250 mM sucrose, 3 mM EDTA) with protease inhibitor. Twenty tightly strokes of homogenizer were performed, followed by 3000 × g centrifugation for 10 min. The supernatant was mixed with 62% sucrose to reach a final sucrose concentration of 42%. Discontinuous sucrose gradient ultracentrifugation was conducted, sucrose gradient were 42%, 35%, 25%, 4 mL for every gradient. Then centrifuge at 200,000 × g overnight. Take 1 mL every time from top to bottom. Mix the 35% sucrose part, diluted 5 times with PBS. This fraction was then incubated with the previously prepared beads overnight. For MS/MS detection, beads were eluted by 1% SDS PBS for 5 min.