Esterase from porcine liver

Reagents were purchased from commercial sources: FITC-labeled 4 kDa, 70 kDa, 150 kDa dextrans (Sigma, USA), TMR 70 kDa-labeled dextran (Life Technologies), TP (Chengdu Purifa, China), succinic anhydride (J&K Scientific, China), DMAP (Macklin, China), CDI (Macklin, China), esterase from porcine liver (Sigma, USA), EIPA (MedChemExpress, USA), and LysoTracker (Life Technologies).
Plasmids were obtained from Addgene (USA): pBABE-Puro-KRAS^G12V was a gift from Christopher Counter and the pBABE-Puro vehicle control was a gift from Hartmut Land, Jay Morgenstern and Bob Weinberg. All plasmids were packaged into lentiviruses with RSV, VSV, and pMDL and transduced into BxPC-3 cells via infection and selected under puromycin pressure (~1.5 µg/ml).
Antibodies used for immunohistochemical staining were from Servicebio (China): anti-Ki67 (GB111141), anti-EpCAM (GB11274), anti-CD3 (GB13014), anti-CD4 (GB13064-2), anti-CD8 (GB13429), anti-CD45 (GB11066), anti-IFN-γ (GB11107-1) and anti-TNF-α (GB11188).

Hexanediol diacrylate (HDD), piperazine (PIP), triethylamine (TEA), dimethyl sulfoxide (DMSO)-d6 (99.5% D atom), chloroform-d (99.8% D atom), 4,4′-azobis(4-cyanovaleric acid) (ACVA, >98%), and esterase from porcine liver were obtained from Sigma-Aldrich without further purification. N-Acryloyl morpholine (NAM) and N,N-dimethylacrylamide (DMA), were purchased from Sigma-Aldrich and the inhibitor removed by passing the monomers through a column of basic aluminium oxide. Acryloxyethyl thiocarbamoyl Rhodamine B was purchased from Polysciences, Inc. Solvents and other reagents were acquired from commercial sources and used as received unless stated otherwise.
2-(((Butylthio)carbonothioyl)thio)propanoic acid (PABTC), N-hydroxysuccinamide-(propanoic acid)yl butyl trithiocarbonate (NHS-PABTC) and (R)-2-(4-(3-(6-chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxypropoxy)phenyl)acetonitrile (QSI61) were synthesised by methods previously reported in literature.^{10,10,26,27 (link)}

Proteins washed from 220 mg of spores were freeze-dried, then dissolved in 500 μL of 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1 mM EDTA, 1% v/v Triton X-100, 0.1 M PMSF, and a protease inhibitor cocktail tablet (Roche Diagnostics). Intracellular proteins were isolated from 220 mg of washed spores by grinding in liquid nitrogen. The tissue powder was dissolved in 500 μL of the same buffer as the extracellular proteins. The lysate was clarified by centrifugation (16,900 × g, 4°C, 20 min), and the supernatant was collected. Protein concentration was determined using the Bradford protein assay. Esterase from porcine liver (Sigma) was used as a positive control of hydrolysis.
Proteins were separated using a Novex Tris-Glycine gel (Thermo Fisher Scientific) and Tris-Glycine Native Sample Buffer (Thermo Fisher Scientific). Preparations containing 1.0, 0.1, or 0.05 μg proteins were loaded onto the gel. SeeBlue Pre-stained Protein Standard (Thermo Fisher Scientific) was used as the molecular weight marker. Electrophoresis was conducted at constant 125 V for 3 h at 4°C in 1× Tris-glycine native running buffer (Thermo Fisher Scientific). The gel was then washed twice for 10 min each in 100 mM Tris-HCI, pH 8.0. Esterase activity was detected using the indoxyl acetate assay as described [50 (link)].