Sec 300

The purity of catalase was examined by a size-exclusion column with a separation range of 10,000 Da–10,00,000 Da BioCore SEC-300) using a high-performance liquid chromatography (HPLC, 1,260 Infinity II system, Agilent) system. Transmission electron microscope (TEM, HT7700, Hitachi Ltd.) was employed to characterize the morphologies of CAT-PEG. Dynamic light scattering (DLS) measurements were performed on a Zetasizer Nano instrument (Malvern Instruments Ltd., United Kingdom) with a 10–mW helium–neon laser and thermoelectric temperature controller. Fluorescent intensity was measured with a Spectra Max M2 plate reader (Molecular Devices). The bioluminescent imaging of the mice was imaged with the IVIS Imaging System (IVIS Spectrum, PerkinElmer). H and E images were taken using inverted fluorescence microscope fluorescence microscope (BK-FL4, ChongQing Optec Instrument Co., Ltd.). TUNEL images were taken using a confocal laser scanning microscopy (Leica TCS SP8). The complete blood count and blood biochemical examination were performed using auto biochemistry analyzer (BS-420, Mindray) and auto hematology analyzer (RJ-0C107223, Mindray), respectively.

SAXS data for unphosphorylated and S241-monophosphorylated PDK1^FL were collected on BM29 at the ESRF, Grenoble, France using an in-line SEC-SAXS setup. Proteins were applied to a Agilent Bio SEC 300 column equilibrated in 20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and 1 % (v/v) glycerol and images were acquired every second for the duration of the size exclusion run. Buffer subtraction was performed by averaging 50 frames on either side of the peak. All subsequent data processing steps were performed using the ATSAS data analysis software 3.9.1. The program DATGNOM^{73 (link)} was used to generate the pair distribution function [P(r)] for each isoform and to determine D_max and R_g from the scattering curves [I(q) vs. q] in an automatic, unbiased manner.