Bca protein assay kit

Purified human and murine CD4⁺ cells were lysed with cell lysis buffer (BioLegend Inc., San Diego, USA), and protein concentration was detected with the BCA Protein Assay Kit (Cell Signaling Technology^®, Danvers, USA). Lysates were run on 4–15% gradient polyacrylamide gels (Bio-Rad Laboratories, Munich, Germany). Blotting was performed with the TransBlot^® Turbo™ Transfer System (Bio-Rad Laboratories). Proteins were detected with the following antibodies: HRP anti-β2-Actin antibody mouse mAb (Abcam, Cambridge, UK), anti-VASP rabbit mAb, and anti-rabbit IgG HRP-linked antibody (Both from Cell Signaling Technology^®, Danvers, USA). Detection was performed by the ImageJ software (NIH, USA).

Purified human CD4+ T cells from RA patients were lysed with cell lysis buffer (BioLegend Inc.). The detection of protein concentration was conducted with the BCA Protein Assay Kit (Cell Signaling Technology®). For gel running, 4%–15% gradient polyacrylamide gels were used (Bio‐Rad Laboratories). Blotting was performed with the TransBlot® Turbo™ Transfer System (Bio‐Rad Laboratories) by using the PVDF membrane. The following antibodies were used for the protein detection: anti‐HEF1/NEDD9 (2G9) mouse mAb (Cat. No. 4044), anti‐mouse IgG HRP‐linked antibody (Cat. No. 7076), and anti‐GAPDH (D16H11) XP® rabbit mAb (HRP conjugate) (Cat. No. 8884) (all from Cell Signaling Technology®). Detection was performed by using ChemiDoc™ MP Imaging System (Bio‐Rad Laboratories), and data were analyzed by the ImageJ software (NIH). PC9 wt cells served as a positive control and PC9^{nedd9−/nedd9–} KO cells as a negative control for NEDD9 expression. PC9 cells are derived from human adenocarcinoma from lung tissue. The cells were kindly provided by Dr. Tamina Seeger‐Nukpezah (University of Cologne).

Human ovarian cancer SKOV3 cells were obtained from Wuhan boster biological engineering co., LTD (Wuhan, China). Cardamonin, rapamycin, 2-deoxy-D-glucose (2-DG) and Compound C (an AMP-activated protein kinase (AMPK) inhibitor) were purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA). The assay kits of ATP, lactate, hexokinase (HK) and lactate dehydrogenase (LDH) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The BCA protein assay kit and antibodies for mTOR, p-mTOR (Ser2448), S6K1, p-S6K1 (Thr389), AMPK, p-AMPK (Ser172), LC3, LAMP1, HK2 and actin were obtained from Cell Signaling Technology (Beverly, MA, USA).

Mouse liver tissue was homogenized in buffer comprising 150 mM NaCl, 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, 50 mM Tris base, 5 mM EDTA, 1 mM EGTA, 10 mM Sodium Fluoride, 1 mM Sodium Orthovanadate, 1 mM PMSF, and Halt^™ Protease and Phosphatase Inhibitor Cocktail (cat# 78440, ThermoFisher Scientific, Waltham, MA). Lysates were subjected to centrifugation at 15,000 x g for 15 min at 4°C to pellet any cell debris, and the protein concentration was determined using the BCA Protein Assay Kit (cat# 7780S, Cell Signaling, Danvers, MA). Samples usually comprising 10–30 μg of protein were mixed with Laemmli sample buffer including 2-mercaptoethanol, heated to boiling for 5 min, and subjected to SDS-PAGE on NuPAGE^™ 4–12% Bis-Tris gels (cat# NP0336, Invitrogen, Waltham, MA) followed by transferring onto PVDF membrane for immunoblotting analysis. Antibodies were raised against CypA as described previously [26 (link), 27 (link)]. Additional antibodies were purchased that were raised against CypB (cat# D1V5J, Cell Signaling, Danvers, MA, USA), KDEL (cat# ADI-SPA-827F, Enzo, Farmingdale, NY, USA), or GAPDH (cat#2118S, Cell Signaling).

Treated gallbladder cancer cells were obtained and then lysed with RIPA Buffer (Beyotime Institute of Biotechnology, China) supplemented with 1 mM Phenylmethanesulfonylfluoride (PMSF). After denaturation at 100°C for 10 min, concentrations of proteins were assessed with the BCA Protein Assay Kit (#7780, Cell Signaling Technology Inc, CST, MA, USA) as described [21 (link)]. The equal total protein (30 μg) amounts were loaded to SDS-PAGE gel (10%) and blotted onto a nitrocellulose (NC) membrane (Millipore, Merck KGaA, Darmstadt, Germany). After blocking with skim milk (5%), the NC membrane was immunoblotted at 4°C in the presence of primary antibodies overnight. Then, the membrane was washed, incubated for 2 h in the presence of a HRP-conjugated secondary antibody. Finally, the protein bands were visualized with the Image Lab system (Bio-Rad Laboratories, Inc.,). Expression levels of GAPDH or beta-actin were used as controls.

Histone acetyltransferase (HAT) activity was measured in nuclear extracts that were prepared using the nuclear extraction kit described above, but without the addition of DTT. The concentration of protein extracts was determined with BCA protein assay kit (Cell Signaling). HAT activity was measured using the Histone Acetyltransferase Activity Assay kit (colorimetric) (Abcam, ab65352) as per the manufacturer’s protocol. The optical density (OD) was measured at 440 nm after 2 hours of incubation and HAT activity was expressed as relative OD_440nm/μg of total protein. Histone deacetylase (HDAC) enzyme activity was measured in nuclear extracts using the HDAC Assay kit (fluorometric) (Active Motif) following the manufacturer’s instructions. Fluorescence was read on a plate reader with 360 nm extraction wavelength and 460 nm emission wavelength. To determine the specific activity of HDAC enzymes, the HDAC standard concentration (μm) was converted into mass (pmol). The enzymatic activity of HDAC in nuclear samples was calculated by dividing the pmol of product formed by the incubation time (minutes) and the mass of HDAC (milligrams) used to yield the activity (pmol/min/mg). The overall enzymatic activity of specific HDACs in all samples, including the positive control (Hela cell nuclear extract) and HDAC inhibitor (Trichostatin A; TSA), was calculated, based on the standard curve.

NHBE cells and lung tissue cells were lysed in lysis buffer (Cell Signaling Technology, Beverly, MA) with protease inhibitor cocktail (Cell Signaling Technology, Beverly, MA and sonicated three times for 2 s each with at least 1-min rest on ice between each 2-s pulse. Samples were centrifuged at 10,000 × g for 5 min at 4°C and the supernatant was collected. Protein concentration was determined by BCA protein assay kit from Cell Signaling Technology.
Thirty micrograms of total protein were mixed in a reducing sample buffer, and then electrophoresed on a 10–15% Tris gel with Tris running buffer, blotted to PVDF membrane, and sequentially probed with primary antibodies against 2′-5′-Oligoadenylate Synthetase 1 (OAS1), Interferon-induced GTP-binding protein Mx1 (MX1), Interferon-stimulated gene 15 (ISG15) (Proteintech Group, Inc. Rosemont, IL). A horseradish peroxidase-conjugated goat anti-rabbit antibody was then added, and secondary antibodies were detected using enhanced chemiluminescence (ECL Plus, General Electric Healthcare, and Milwaukee, WI).