Mammalian protein extraction reagent

Testis tissue, cells, and exosome fractions were lysed in Mammalian Protein Extraction Reagent (78501, Thermo Scientific) containing a protease inhibitor cocktail. The proteins were separated by 8–12% bisacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The proteins were reacted with primary antibodies followed by a horseradish peroxidase (HRP)-conjugated secondary antibody. Immunodetection was carried out with Chemiluminescent HRP substrate. Normalization was conducted by blotting the same samples with an antibody against β-actin or GAPDH.

On day 7, protein was extracted from cells using Mammalian Protein Extraction Reagent (Thermo Scientific) according to the manufacturer’s instructions. Protein samples were resolved using the iBlot 2 Dry Blotting System (Life Technologies, Carlsbad, CA, USA). Membranes were treated with the following primary antibodies: anti-NF-ATc1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-β3-integrin (Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH (Santa Cruz Biotechnology), and anti-citrullinated fibrinogen (ModiQuest Research). The membranes were then treated with the corresponding secondary antibodies. Detection was performed using the Luminata Forte Western HRP Substrate (Millipore).

Parasitized erythrocytes were transferred to RPMI1640 medium supplemented with 25% fetal bovine serum, 0.05 mg/mL penicillin and 0.05 mg/mL streptomycin. The parasitized erythrocytes were incubated for 22 h in 90% N₂, 5% CO₂ and 5% O₂. Mature schizonts and gametocytes were harvested by Nycodenz density gradient centrifugation, as described previously [7 (link)]. Proteins were extracted using Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. Protein IP in transgenic parasites expressing mCherry fused to PBANKA_1019700 was performed using RFP-Trap Agarose and a RFP-Trap-A kit, according to the manufacturer’s instructions (Chromotek, Planegg, Germany).

Total cell protein was extracted using Mammalian Protein Extraction Reagent (Thermo Fischer Scientific), supplemented with 1% Halt Protease Inhibitor Cocktail (Pierce Biotechnology, Rockford, IL, USA). The supernatants were collected after centrifugation at 15,000 rpm at 4 °C for 5 min. Protein concentration was measured by Protein Assay Rapid Kit (Wako), according to the manufacturer’s instructions. Capillary electrophoresis immunoassay for detection of PD-L1 protein was employed by using Simple Western System Wes (ProteinSimple, California, USA). Protein samples and reagents (EZ Standard Pack 1, ProteinSimple) were loaded into the assay plate. The protein (ng/mL) was electrophoresed in capillary, which was filled with a stacking and a separation matrix (Jess/Wes 25-Capillary Cartridge, ProteinSimple). The proteins separated by the photoreactive binding reaction were immobilized on the inner wall of the capillary. Primary antibodies were as follows: Anti-PD-L1 XP monoclonal antibody (E1L3N, Cell Signaling Technologies, Danvers, MA, USA) at 1:100, or anti-β-actin monoclonal antibody (Sigma–Aldrich) at 1:1000. The target proteins were immunodetected with HRP-labeled secondary antibody and a chemiluminescent substrate (ProteinSimple). The data were analyzed using Compass software (ProteinSimple).

Breast tumors harvested for kinome/MIB-assay studies were homogenized before protein extraction. Additionally, protein extracts were collected from 67NR cells (1.5x10⁶) grown for 24 hours before treatment with AEE788 (1 μM), ATX-101 (6 μM) or the combination of these for additional 24 hours (n=3 for each treatment group). Proteins were extracted using Mammalian Protein Extraction Reagent (Thermo Fischer Scientific) according to the manufacturer’s instructions and with Halt Protease inhibitor cocktail (EDTA free) and Halt Phosphatase inhibitor cocktail (Thermo Fischer Scientific) added. The final protein concentrations were adjusted to 1 mg/mL.
The MIB-assay enriching for kinases was performed using three different kinase inhibitors (Purvalanol B (Tocris Bioscience), Bisindolylmaleimide X (Activate Scientific) and SB6-060-05 [49 (link)]) immobilized on ECH sepharose 4B and EAH sepharose 4B beads (GE healthcare) as described [29 (link)], using 200 μL protein extract (1 mg/mL) per column with beads. Proteins in the eluates were identified using mass spectrometry (MS)-analysis (Orbitrap) as described [29 (link)].

Cells were lysed in MPER™ (Mammalian Protein Extraction Reagent, Thermo Scientific, Rockford, IL), which was supplemented with cOmplete Protease Inhibitor Cocktail tablets and PhosSTOP Phosphatase Inhibitor Cocktail tablets (Roche, Indianapolis, IN). Protein concentrations of lysates were determined using the BCA assay (Thermo Scientific, Rockford, IL) and proteins were resolved on SDS-PAGE gels and transferred to PVDF membrane (EMD Millipore, Billerica, MA) using standard procedures. Membranes were blocked with SuperBlock™ T20 (TBS) Blocking Buffer (Thermo Scientific, Rockford, IL) and incubated overnight at 4°C in primary antibodies of Runx2 (Cell Signaling, #8486, 1:1000), ALP (Abcam, #ab67228, 1:1000), RANKL (Cell Signaling, #4816, 1:1000), Osteocalcin (Santa Cruz, #sc-30044, 1:500), Osterix (Abcam, #ab22552, 1:500), and β-actin (Sigma-Aldrich, #A1978, 1:5000). Membranes were then incubated with either anti-mouse (Cell Signaling, #7076, 1:2000) or anti-rabbit IgG (Cell Signaling, #7074, 1:2000), HRP-linked secondary antibodies and Chemiluminescented by Pierce™ ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL). Quantification of signals on Western blots was done using National Institutes of Health (NIH) ImageJ Imaging and Processing Analysis Software with signaling intensity normalized to β-actin.