Origin 9.1 pro

The optical time-lapse images were processed with the Zen 2.1 black software by Zeiss, followed by drift corrections, region of interest analysis, and mean grey value extraction by ImageJ. The monoexponetial translocation kinetics were fitted with the Nanocal software (Nanospot GmbH), and the resulting fist-order rate constants (k_efflux) were statistically analysed with Origin 9.1 Pro (OriginLab), including Gaussian fitting. The antibody-sink reaction was analysed in a similar manner; however, the onset of the EGFP increase traces was fitted by a linear function due to the constant flux gradient mediated by the antibodies and due to the similarity in the efflux rate.

Silk cocoons were degummed by boiling a solution of Na₂CO₃, according to a previously published method (Liang et al., 2020 (link)). The resultant degummed silk fibers were observed using scanning electron microscopy (SEM; TM4000Plus, HITACHI, Japan) and transmission electron microscopy (TEM; Talos F200X, Thermo Scientific, United States). Prior to the SEM observation, the specimens were sputter-coated with gold to achieve high conductivity. For TEM observation, the degummed silk fibers were cut into fine pieces and sonicated in ethanol for 10 min. A drop of the dispersion was subsequently placed on a carbon-coated copper grid and dried in an oven at 60°C.
Samples were prepared for FTIR analysis following the KBr pellet method (Liang et al., 2020 (link)). Each sample was scanned 32 times, from 400 cm⁻¹ to 4,000 cm⁻¹, with a resolution of 4 cm⁻¹. Quantitative analysis of the protein secondary structure was performed for the amide I band from 1,600 cm⁻¹ to 1,720 cm⁻¹ using a Fourier-transform infrared (FTIR) spectrometer (Nicolet iN10, Thermo Scientific, United States). The baseline of the amide I band was first corrected, and the peak was fitted with a Gaussian function using Origin 9.1 Pro software (Origin Lab, United States).