Pierce bicinchoninic acid bca assay

The cells were treated under the indicated conditions and then efficient lysed with RIPA buffer at pH 8.0 (150 mM NaCl, 50 mM Tris, 1% Triton X‐100, 0.5% sodium deoxycholate, 0.1% SDS). All the samples were quantified using Pierce^TM bicinchoninic acid (BCA) assay (Thermo Fisher Scientific Inc.) to ensure equal loading of proteins. The cellular protein samples were separated by SDS‐PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore). After blocked by 5% BSA, the membrane was probed with the primary antibody and then incubated with HRP‐conjugated secondary antibody in TBST. The primary antibodies include anti‐TXN2 (1:1000 dilution, Abcam, ab185544) and anti‐HP (1:1000 dilution, Abcam, ab256454).

Confluent cells were lysed in 1X radioimmunoprecipitation assay (RIPA) buffer, centrifuged at 14,000 × g for 10 min, and supernatants were stored at −80°C prior to Western blot analysis. Protein concentration was determined using a PierceTM bicinchoninic acid (BCA) assay (ThermoFisher), and xxμg protein from each cell lysate was separated by SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) and transferred onto a methanol-activated PVDF membrane (Millipore). The membrane was blocked in 5% BSA for 1 h at room temperature and incubated at 4°C overnight with primary antibody against βIII tubulin (1:1,000 (0.5 μg/mL), R&D Systems MAB1195), STAT3 (1:1,000 (0.05 μg/mL), Cell Signaling Technology mAb No. 9139), p-STAT3 (1:2000 (0.05 μg/mL), Cell Signalling Technology mAb No. 9145) or GAPDH (1:1,000 (0.2 μg/mL), Santa Cruz Biotechnology sc-47724). After overnight incubation, the membrane was washed in 0.1% TBS-Tween (TBS-T) and incubated at room temperature for 1 h with goat anti-rabbit secondary antibody (1:5,000, Cell Signaling Technology mAb No. 7074) or HRP-conjugated mouse IgG_κ light chain binding protein (1:2000 Santa Cruz Biotechnology Product No. sc-516102). Membrane was washed in TBS-T and developed using Pierce™ ECL Western Blotting Substrate (Thermo Scientific) and the Fujifilm LAS3000 luminescent image analyzer.

MM1.S human MM 63 (link) and 5TGM1 murine MM 64 (link) cell lines, either naïve or carrying Click Beetle Red (CBR) luciferase and Green Fluorescent Protein (GFP) reporters (MM1.S/CBR/GFP and 5TGM/CBR/GFP, respectively), as well as MM1.S/CBR/GFP resistant to RaST and MM1.S/CBR/GFP without CD49d, were generously provided by Dr. DiPersio (Washington University School of Medicine, WUSM, St. Louis). Cells were routinely cultured in complete medium (CM) consisting of Iscove's Modified Dulbecco's Media (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, ThermoFisher Scientific, Waltham, MA) and 50 µg/mL Gentamycin (Thermo Fisher Scientific, Waltham, MA). The cells were routinely washed in phosphate-buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA), pH 7.4. VLA-4 and αvß3 targeted NM were loaded with TC (Bis(cyclopentadienyl)titanium(IV) dichloride, Sigma-Aldrich, St. Louis, MO) 43 , 46 (link). Protein content was routinely measured with Pierce^TM bicinchoninic acid (BCA) assay (Thermo Fisher Scientific Waltham, MA).