Cm5 chip

Chemicals were obtained from Sigma if not stated otherwise. Single-fluorophore, 5′-end-labeled RNA fragments from mouse β-actin mRNA (accession number: NM_007393.5) and mouse β2-tubulin-mRNA (accession number: NM_023716.2) were purchased from IBA-Lifesciences (Germany). All 25 nt RNAs used for SPR experiments were purchased from IDT. The fragments for β-actin and β2tub mRNAs were taken from the same transcripts listed above. The Map1b-fragment comes from accession number NM_008634.2, the Camk2α-mRNA fragment from NM_001286809.1. TIRF-M experiments were performed on an iMIC (TILL Photonics, Germany) TIRF microscope equipped with three Evolve 512 EMCCD cameras (Photometrics, UK), a 100× 1.49 NA objective lens (Olympus, Japan), a quadband filter (405/488/561/647, Semrock, USA), and four different laser lines (405 nm, 488 nm, 561 nm, and 639 nm). An Olympus tube lens adds a post magnification of ×1.33, which results in a final pixel size of 120.3 nm. SPR experiments were performed on a BIACORE T100 system (GE Healthcare) using the Twin-Strep-tag® Capture Kit (IBA-Lifesciences) and CM5-Chips (Cytiva).

The method of SPR was used as previously described (53 (link)) using a Biacore 8K instrument with CM5 chips (Cytiva) at room temperature. The physical absorption response signal of PI3Kα on CM5 chips with four different pH buffers (4.0, 4.5, 5.0, and 5.5) was tested to find the optimal buffer. PI3Kα (1 mg/mL) was diluted to 20 μg/mL with acetate buffer (pH 5.5) and immobilized through a standard amine-coupling protocol with an amine coupling kit (Cytiva). In order to obtain the best-fitting kinetics model, these compounds were serially diluted with Biacore HBS-EP+ buffer (Cytiva) to different concentrations, respectively. The analytes passed through chip surface flow cell 1 (fc1) and flow cell 2 (fc2) at a rate of 30 μL/min. An association phase of 120 s was followed by a dissociation phase of 240 s. The association and dissociation curves of the sensorgrams were corrected for obtaining signals in the reference flow channel (fc1), which is a control without PI3Kα, and then analyzed with the Biacore Insight Evaluation (v2.0.15.12933). The affinity of an interaction can be derived from a steady state measurement, which represents the K_D values obtained by fitting the binding curve with Biacore Insight Evaluation (v2.0.15.12933). The SPR data were presented as the mean value, calculated from at least three independent measurements per compound.

The apparent affinity of HIV-1 bNAbs (IgG and Fabs) to KLH, YU-2, and ZM-96 gp140 trimers, CM5 chips (Biacore) were measured as previously described (Mouquet et al., 2010 (link)). Thermodynamic parameters were determined using Eyring’s analyses as previously described (Hadzhieva et al., 2017 (link)). See the Supplemental Experimental Procedures for further details.