Pierce bca protein assay

For stimulation with recombinant EGF (10 ng/ml) (GIBCO, Thermo Scientific, MA, USA), and/or erlotinib inhibition (conc. as indicated) up to 24 h, SCaBER, J82, and HT1376 cells were cultured in serum-free media, containing human transferrin and 1% DMSO (Sigma-Aldrich). For p-SCC cells standard conditions (see Supplementary Information) were used. Cellular proteins were extracted in RIPA lysis buffer containing phosphatase inhibitors and quantified using the Pierce^TM BCA protein assay (Thermo Scientific, MA, USA). RNA was extracted using the Nucleospin RNA Plus Kit.

For protein extraction, cultured cells were lysed in RIPA lysis buffer (Cell Signaling, Danvers, MA) with freshly added PMSF, sodium orthovanadate and complete protease inhibitors [23 (link)]. Protein concentration was determined with the Pierce^TM BCA protein assay (ThermoFisher, Philadelphia, PA). Immunoblotting was done with anti-IGF2 antibody (Biorbyt, Cambridge, UK) according to the manufacturer’s protocol. Briefly, 4–12% Bis-Tris polyacrylamide gels (NuPage, Invitrogen) were equally loaded with 5–30μg of protein, electrophoresed at 140-200V, and transferred to nitrocellulose membrane by iBlot dry transfer. Membranes were blocked with either 5% BSA or LI-COR TBS blocking buffer for 30 minutes to 1 hr. The membrane was probed with primary antibodies to IGF2 (Biorbyt, Cambridge, UK) and pSTAT3, STAT3, TSC2, pS6, S6, GAPDH and β-actin (all from Cell Signaling Technology, Inc., Beverly, MA) at 4°C overnight. Appropriate secondary antibodies were incubated for 1 hr at room temperature. Blots were developed either by ECL prime Western blotting detection reagents (GE—Amersham Bioscience, Piscataway, NJ) or by secondary fluorescent antibodies (LI-COR, Lincoln, NE). Images were captured using a LI-COR Odyssey-Fc imager with LI-COR advanced imaging software.

According to the manufacturer’s instructions, the protein content of the obtained plasma sEV preparations was analyzed using Pierce^TM BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA). The total protein of sEVs was determined directly after sEV preparation by size-exclusion chromatography (unconcentrated samples). For Western blots or bead-bound flow cytometry, sEVs were concentrated on centrifugal filter units (100 kDa, Merck Millipore, Burlington, MA, USA) and the protein amount was detected by BCA.

Protein extraction was done with TritonX-containing lysis buffer and protein content was determined using BCA assay (Pierce^TM BCA protein assay, Thermo Fisher Scientific). SDS–PAGE was carried out followed by semidry blotting. After blocking the membrane for 1 h with 5% milk, proteins were detected using the following antibodies: rabbit anti-MCL-1 (Enzo, ADI-AAP-240F), mouse anti-BCL-2 (Dako (Agilent), M088701-2), rabbit anti-BCL-x_L (CellSignaling, 2762 S), mouse anti-NOXA (Enzo, ALX-804-408), mouse anti-BAX (BD Bioscience, 610983), rabbit anti-BAK (Upstate/ Merck, 06–536), rabbit anti-BIM (CellSignaling, 3183 S) and rabbit anti-caspase-3 (CellSignaling, 9662 S), mouse anti-β-Actin (Sigma, A5441), mouse anti-GAPDH (BioTrend (Hy Test Ltd), 5G4-6C5) or mouse anti-Vinculin (Sigma/Merck, V9131-100UL). Quantification of protein expression was performed using ImageJ 3.1 software.

100 µL M-PER lysis buffer (Mammalian Extraction Buffer, #78503, #78420, #87785, ThermoFisher Scientific^TM, Darmstadt, Germany) was added per 1 × 10⁶ cells and syringe-and-needle homogenization was performed. The lysates were incubated for 1 hour on ice, centrifuged for 15 min at 16.000 × g at 4 °C. Next, the supernatant (containing the proteins) was transferred to pre-cooled clean tubes. Aliquots of 10 µL sample volume were prepared and snap-frozen by liquid nitrogen before storage at −80 °C. Protein quantification was conducted by bicinchoninic acid assay according to the manufacturer’s instructions using Pierce^TM BCA Protein Assay (#23225, Thermo Fisher). Protein concentrations were measured using microplate reader TECAN Infinite®200 PRO (Tecan Trading AG, Männedorf, Switzerland).

To determine intracellular cholesterol levels, a Cholesterol/Cholesterol Ester-Glo^TM Assay (Promega) was performed according to the manufacturer’s instructions. In brief, 20,000 cells were seeded per well in a 96-well plate in RPMI + 10% CS FCS. After 72 h of treatment, medium was removed and the cells were lysed with 50 µL Cholesterol Lysis Solution per well for 30 min at 37 °C. The cell lysis solution was then transferred into another 96-well plate (Corning 3610, white, clear bottom) and mixed with Cholesterol Detection Reagent in the presence of esterase. After incubation for 1 h at room temperature, chemoluminescence was measured on a Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek Instruments). Total cholesterol levels were normalized to protein content that was determined by Pierce^TM BCA protein assay (Thermo Fisher Scientific) and expressed as µM cholesterol.