Microcal calorimeter

ITC experiments were performed on a PEAQ-ITC Microcal calorimeter (Malvern) at 25°C. Protein samples (in 50 mM Tris-Cl, pH 7.5, 100 mM NaCl) were prepared for titrations (13 or 19 titrations in total for each measurement). PINCH1_LIM45C or LIM5C and corresponding mutants with the concentration of 200 μM in the syringe were injected into the sample well containing 20 μM Rsu1 protein with the titration speed 0.5 μl/s. A time interval of 150 s between two titration points was applied to ensure that the titration peak returned to the baseline. The titration data were processed by MicroCal PEAQ-ITC Analysis Software and fitted by the one-site binding model.

ITC experiments were performed on a PEAQ-ITC Microcal calorimeter (Malvern) at 25 °C. Protein samples (in 50 mM Tris-Cl, pH 7.5, 100 mM NaCl) were prepared for titrations. Each measurement included 13 titrations. In total, 400 μM DLC-1 was injected into the sample well containing 40 μM SAO-1 protein with a titration speed of 0.5 ml/s.
A time interval of 150 s between injections was used to ensure that the titration peak returned to the baseline. The titration data were processed by MicroCal PEAQ-ITC analysis software and fit by the one-site binding model.

ITC measurements were carried out on a VP-ITC MicroCal calorimeter (Malvern) at 25 °C. All proteins were dissolved in the buffer containing 50 mM Tris, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT at pH 7.8. Nfasc proteins (200 μM) were loaded into the syringe, and AnkG proteins (20 μM) were loaded in the cell. Each titration point was obtained by injecting a 10 μl aliquot of syringe protein into the cell at a time interval of 180 s to ensure that the titration peak returned to the baseline. The titration data were analyzed using the program Origin 7.0 (Microcal) and fitted by the one-site binding model to determine the binding affinities of Nfasc fragments with ANK repeats.

ITC measurements were carried out on a VP-ITC Microcal calorimeter (Malvern) at 25 °C. All proteins were in 50 mM Tris buffer containing 100 mM NaCl, 1 mM EDTA, and 1 mM DTT at pH 7.8. Each titration point was performed by injecting a 10 μL aliquot of γ2 subunit protein or peptide (100–200 μM) into GABARAP, GABARAPL1, or AP2 protein samples (10–20 μM) in the cell at a time interval of 180 s to ensure that the titration peak returned to the baseline. The titration data were analyzed using the program Origin7.0 and fitted by the one-site binding model.

ITC experiments were performed on a VP‐ITC Microcal calorimeter (Malvern) at 25 °C. Titration buffer contained 50 mm Tris/HCl (pH 7.5), 100 mm NaCl, 1 mm DTT, and 1 mm EDTA. For a typical experiment, each titration point was obtained by injecting a 10 μL aliquot of GABARAP or LC3A sample (350 μm) into the cell containing Trx‐tagged FAM134B (35 μm) at a time interval of 120 s to ensure that the titration peak returned to the baseline. The titration data were analyzed using the one‐site binding model by Origin7.0.

ITC experiments were carried out on a VP-ITC Microcal calorimeter (Malvern) at 25 °C. All proteins were dissolved in buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT. Each titration point typically is consisted of injecting 10 μl aliquots of the liprin-α fragments or their mutants, at concentration of 200 μM into the solution of containing LAR_D1D2, its mutants, LAR_D2, PTP-σ_D1D2, or PTP-δ_D1D2 at a concentration of 20 μM. A time interval of 150 or 180 s between two titration points was used to ensure the complete equilibrium of each titration reaction. The titration data were analyzed using the program Origin7.0 and fitted by a one-site binding model.