Goat anti rabbit hrp secondary antibody

Halofuginone hydrobromide, RNase A, 5-Fluorouracil, puromycin, and Pierce (R) BCA Protein Assay Kit were obtained from Sigma-Aldrich (Munich, Germany). Artemisinin was purchased from Abcam (Cambridge, UK). Antibodies against p21^Waf1/Cip1 (12D1), p27^Kip1 (D69C12), phospho-CDK2 (Thr160), and β-Actin were purchased form Cell Signaling Technology (Danvers, MA). HRP-goat anti-rabbit secondary antibody was purchased from Invitrogen (Carlsbad, USA). Goat anti-mouse IgG-HRP secondary antibody was purchased from San Cruz Biotechnology (Santa Cruz, USA).

After cells were treated with HF for 12 h, whole cell lysates were obtained by suspending the cells in lysis buffer. Followed by centrifugation at 13,500 rpm for 15 min at 4°C, total protein concentration was measured using Pierce(R) BCA Protein Assay Kit and 10 to 25 μg of protein was separated on 10% sodium dodecylsulphate-polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes. After blocking (5% skim milk powder in TBST, 20) for 1 h at room temperature, the membrane was then incubated with primary antibody overnight at 4°C. Antibodies against VDAC (D73D12), phosphor-mTOR (Ser2448), phosphor-p70S6 Kinase (Thr389), phosphor-4EBP1 (Thr37/46), phosphor-Akt (Ser473), Akt (pan) (C67E7), 4EBP1, p70S6 Kinase (49D7), mTOR (7C10), cleaved-PARP, cleaved-caspase 3, GLUT1 (D3J3A), fatty acid synthase (C20G5) and β-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA). The membrane was incubated with secondary antibody for 1 h at room temperature. HRP-goat anti-rabbit secondary antibody was purchased from Invitrogen (Carlsbad, USA). Goat anti-mouse IgG-HRP secondary antibody was purchased from San Cruz Biotechnology (Santa Cruz, USA). All antibodies were diluted in TBS-Tween 20 containing 5% dry milk. The immune-reactive proteins were detected by enhanced chemiluminescence (ECL) using X-ray film and ECL reagent.

Halofuginonehy drobromide, 5-fluorouracil, Earle's Balanced Salt Solution (EBSS), chloroquine (CQ), propidium iodide (PI) and Pierce (R) BCA Protein Assay Kit were obtained from Sigma-Aldrich (Munich, Germany). Artemisinin and In situ BrdU-red DNA fragmentation (TUNEL) assay kit were purchased from Abcam (Cambridge, UK). Antibodies against cleaved capase-8, cleaved capase-9, cleaved caspase-3, cleaved PARP, SQSTM1/p62, LC3-II and β-Actin were purchased form Cell Signaling Technology (Danvers, MA, USA). HRP-goat anti-rabbit secondary antibody was purchased from Invitrogen (Carlsbad, CA, USA). Goat antimouse IgG-HRP secondary antibody and caspase-3 inhibitor (z-DEVD-fmk) were purchased from San Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-8 inhibitor (z-IETD-fmk) and caspase-9 inhibitor (z-LEHD-fmk) were purchased from R&D Systems (Wiesbaden-Nordenstedt, Germany). FITC Annexin V Apoptosis Detection Kit I was obtained from BD Bioscience (San Jose, CA, USA).

Proteins were extracted from HCC cells with RIPA lysis buffer, followed by centrifugation at 13,500 rpm for 15 min at 4 °C. Protein concentration was measured using Pierce (R) BCA Protein Assay Kit, and equal amount of protein was separated on 10% SDS-PAGE and transferred to PVDF membranes. After blocking (5% skim milk powder in TBS-Tween 20) for 1 h at room temperature, the membrane was then incubated with the respective primary antibody overnight at 4 °C. Afterward, the membrane was incubated with secondary antibody for 1 h at room temperature. All antibodies were diluted in TBS-Tween 20 containing 5% dry milk. The immune-reactive proteins were detected by enhanced chemiluminescence (ECL) using X-ray film and ECL reagent. The primary antibodies used were the following: porimin (Santa Cruz, 377189), ERO1 (Santa Cruz, 365526), PERK (Santa Cruz, 377400), p-PERK (Beyotime, AF5902), ATF6α (Santa Cruz, 22799), IRE1α (CST, 3294), p-eIF2α (CST, 9721), eIF2α (CST, 9722), ATF4 (Santa Cruz, 390063), CHOP (Santa Cruz, 7351), and GAPDH (Santa Cruz, 365062). HRP-goat anti-rabbit secondary antibody (Invitrogen, 32460) and HRP-goat anti-mouse secondary antibody (Invitrogen, 31430) were used.

The cells were washed with PBS and lysed in Laemmle sample buffer (200 mM Tris-HCl, pH 6.8, 8% sodium dodecyl sulfate, 0.4% Bromophenol blue, 20% glycerol, 5% 2-mercaptoethanl). The lysates were sonicated and heated at 95°C for 10 min. The protein samples were loaded and separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (Bio-Rad, Cat#1620112). Blots were then blocked with 5% skim milk in PBST (PBS containing 0.2% Tween-20) for at least 2–3 h at room temperature and Immunoblotting was performed overnight at 4°C with the following antibodies: NAPB antibody (Abcam ab228771, 1:1000), GAPDH (Abcam: 1:2000) The blots were washed the next day and incubated with Goat anti-Rabbit-HRP secondary antibody (Cat# 31460, Thermo Fisher Scientific, 1:3000). The Protein bands were subsequently scanned using the ChemiDoc imaging system (BioRad).