Femtolucent plus hrp

Total protein lysates from tumor cells or tissue were prepared by sonication in lysis buffer (iNtRON). Equal amounts of lysates were boiled at 100°C and resolved using 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) that were blocked with 5% milk in tris-buffered saline and Tween 20 and incubated overnight with the primary antibody, and proteins were detected with an horseradish peroxidase (HRP)-conjugated antibody (Cell Signaling Technology). Membranes were developed by the enhanced chemiluminescence (ECL) method using femtoLUCENT™ PLUS-HRP (G-Biosciences, St. Louis, MO, USA)

For immunoprecipitations, extracts were prepared by cell lysis in immunoprecipitation buffer (50 mM HEPES, pH 7.2; 150 mM NaCl; 1 % NP-40; 1 mM EDTA; 1:100 dilution of protease inhibitor cocktail set III; 1:50 dilution of phosphatase inhibitor cocktail set V; and 0.5 mM dithiothreitol). Cleared lysates were incubated with antibody for 16 h, and subsequently with protein A/G-agarose beads (Thermo Fisher Scientific) for 2 h, at 4° C, on a rotator. Immunocomplexes were washed 3 times with immunoprecipitation buffer and eluted bound-proteins were resolved in 8 or 12 % SDS-polyacrylamide gels. For other experiments, extracts were prepared by cell lysis in RIPA buffer (50 mM HEPES, pH 7.2; 150 mM NaCl; 1 % NP-40; 0.5 % sodium deoxycholate; 0.1 % SDS; 1 mM EDTA; 1:100 dilution of protease inhibitor cocktail set III, 1 mM orthovanadate; 5 mM NaF; and 1 mM dithiothreitol). Following electrophoresis, proteins were transferred onto a PVDF membrane. Blocking and incubation with primary antibodies were performed in Tris-buffered saline containing 0.1 % Tween-20 and supplemented with 5 % skimmed milk powder. Proteins were visualized by ChemiDoc XRC+ gel imaging system (Bio-Rad, CA, USA), using secondary horseradish peroxidase-conjugated antibodies and enhanced chemiluminescence reagent (femtoLucent plus-HRP, G-Biosciences, MO, USA).

The protein was extracted from the cell and tissue lysates (buffer composition: HEPES 200 nM pH-7.4, sucrose 250 mM, KCl 10 mM, MgCl2 1.5 mM, EGTA 1 mM pH-7.4, EDTA 1 mM pH-7.4, DTT 1 mM, PMSF, 1 mM, nonidet P40 0.05%, protease inhibitor cocktail and pepstatin A 1 mM), as reported previously, and immunoblotting was performed [39 (link)] using primary antibodies with appropriate secondary antibodies. The immunoblots were visualized using ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA) after developing the signal with substrate Femto Lucent plus HRP (G-Biosciences). The mean intensity (intensity/area) of bands was determined and normalized against β-actin using ImageJ software (NIH, USA).

The protein samples for analysis were run on 12% of SDS-PAGE for RBD and on 8% for the spike pre-fusion trimer (S2P). The gel was run for 2 h at 120 Volts for the proper resolution of protein. Resolved proteins in gel were then transferred to a PVDF membrane. The membrane was blocked with 5% skimmed milk, incubated with primary Ab (1:5000; pooled sera from RBD immunized groups, SARS/SARS-CoV-2 coronavirus spike protein (subunit 1) polyclonal antibody; 1:1000, anti His antibody; 1:1000), incubated overnight. The membrane was incubated with HRP conjugated anti-mouse (1:2000 for anti RBD sera and anti His blots) and anti-rabbit secondary (1:2000 for SARS polyclonal sera) antibodies and developed using Femtolucent Plus HRP (G Biosciences).

Cells were appropriately treated, and total cell lysates and nuclear extracts were prepared as previously described [35 (link)]. Total protein lysates from cells were prepared by sonicating cells in lysis buffer (iNtRON, Korea). Equal amounts of lysates were boiled at 100°C and resolved by 10-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) and then blocked with 5% milk in Tris-buffered saline and 0.1% Tween 20. The membranes were incubated overnight with primary Abs against LC3, AMPKα, phosphorylated AMPKα, p70S6K, phosphorylated p70S6K, and β-actin (all from Cell Signaling Technology, Danvers, MA), and proteins were detected with goat-anti-rabbit Abs conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, Danvers, MA). Proteins were detected using the enhanced chemiluminescence (ECL) method with femtoLUCENT ™ PLUS-HRP (G-biosciences, St. Louis, MO).

Western blotting reagents, including buffers and gels, were obtained from Bio-Rad Laboratories (Hercules, CA, USA). NP-40 lysis buffer and 4x Laemmle sample buffer were purchased from Alfa Aesar (Haverhill, MA, USA). ThioS, bovine serum albumin (BSA), and donkey serum were purchased from Sigma-Aldrich (St. Louis, MO, USA). Non-fat dry milk was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Protease inhibitor and FemtoLUCENT™ PLUS-HRP were obtained from G-Biosciences (St. Louis, MO, USA). Pierce ECL WB substrate was purchased from Thermo-Fisher Scientific (Waltham, MA, USA). All other chemicals were purchased from VWR (Radnor, PA, USA) or Fisher Scientific (Hampton, NH, USA). Antibodies used for Western blot (WB) and IHC are summarized in Table 1.