Micro bca assay

FMRP quantification closely followed the homogeneous time-resolved fluorescence protocol described in Kim et al.^{13 (link)} The protocol utilizes the Cisbio Human FMRP assay kit. Total protein concentrations were determined using the Thermo Fisher Micro BCA Assay (Thermo Fisher; Waltham, MA; cat. no. 23235) and were used to determine the ratio of interplated FMRP to total protein.

T cells were washed and resuspended in warm CTL medium. T cells were incubated with control, SCT, SCT/CD28 or ROR1 beads in a 37°C water bath at a final cell concentration of 2×10⁷ cells per mL and a bead to cell ratio of 7.5μL per million cells. In some experiments, bi-specific T cells were incubated with K562/B8, K562/B8/EBV, and K562/B8/ROR1 tumor cells at a T cell to tumor cell ratio of 4:1. After the allotted time, cells were quickly washed twice using ice-cold PBS, then lysed in either a 6M Urea, 25mM Tris (pH 8.0), 1mM EDTA, 1mM EGTA solution for mass spectrometry assays or NP40 Cell Lysis Buffer (Thermo Fisher) for immunoprecipitations. Both lysis buffer solutions were supplemented with protease (Sigma) and phosphatase inhibitors (Sigma) at a 1:100 dilution prior to use. When using the urea-based lysis buffer, lysates were sonicated for 15 seconds immediately. When using the NP40 Cell Lysis Buffer, lysates were incubated on ice for 15 minutes with gently mixing every 5 minutes. After sonication or incubation, lysates were cleared by centrifugation at 10,000g and 4°C for 10 minutes. Beads were removed during lysate clearing and protein concentration was quantified by BCA Assay or Micro BCA Assay (ThermoFisher).

PANC-1 and BxPC-3 cells were seeded onto 6-well dishes and incubated for 24 hr in one of the following treatment groups: no treatment, vehicle, free FdUMP, mPEG-FdUMP-CPSNPs, and mPEG-CPSNPs. Lysates were collected by aspirating the media, washing with 1x PBS, and adding RIPA buffer containing Complete Mini protease cocktail (Roche). Protein concentration was determined by microBCA assay (Thermo Scientific) and 20 μg of total protein was separated by gel electrophoresis. After transfer to HyBond ECL and blocking for 1 hr in 5% BSA, blots were probed overnight with anti-TS antibody (#9045 Cell Signaling Technology, 1:1000) or beta-actin antibody (#A2228; Sigma, 1:10,000). Membranes were washed, incubated with secondary antibody coupled to horseradish peroxidase (Amersham), and visualized using an enhanced chemiluminescent substrate (Pierce). Quantitation of scanned blots was done using Image-J software (NIH). After normalizing to β-actin, % active TS is the amount of uncomplexed TS divided by the amount of total TS (active TS and TS-FdUMP ternary complex).

A modified MicroBCA assay (Thermo Scientific) was performed to verify
the catechol-assisted Cu(I) production from Cu(II). This assay relies on the
reduction of Cu(II) ions into Cu(I) ions by chemically reductive samples in an
alkaline buffer (kit component A), which complexes with bicinchoninic acid (BCA,
kit component B) to give a characteristic violet color. CuSO₄ (5 mM),
THPTA (10 mM), p-DOPAmide (10 mM), and S3 (10 mM) were
selectively mixed to provide testing solutions. These solutions were mixed with
MicroBCA kit components A and B at a ratio of 1:5:5. The resulting mixtures were
shaken at 37 °C for 30 min to facilitate coloration, and the absorbance
was measured at 570 nm.