Chemidoc xrs image system

As previously described, total RNAs were extracted from transfected K562 cells using the QIAzol reagent (Qiagen GmbH, Hilden, Germany) [11 (link)]. RNA was quantified by spectrophotometer analysis, and DNA contamination was excluded using a MOPS-buffered 1.5% agarose gel containing formaldehyde as a denaturing agent. After gel electrophoresis in MOPS 1× buffer (20 mM MOPS pH 7.0, 8 mM Sodium Acetate, 1 mM EDTA pH 8.0), the integrity of RNA was confirmed by UV exposure using a ChemiDoc XRS Image System (Bio-Rad Laboratories), according with the manufacturer’s instructions.

MDSCs were harvested in RIPA buffer with protease inhibitor tablets and sonicated on ice. The lysate was then centrifuged at 14,000 rpm at 4°C for 15 minutes, and the supernatant was reserved. Protein lysates were denatured in the SDS sample buffer at 100 °C for 5 minutes and then were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, USA). After blocking in 5% milk, the membrane was incubated with a primary antibody at 4°C for overnight. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 hour. Detection and analysis were performed using the Chemidoc XRS Image system and Image Lab 5.0 software (BioRad). For immunoprecipitation, whole-cell lysates were incubated with 1 μg of antibody and protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. The eluted immunoprecipitates were resolved via SDS-PAGE, and the associations between proteins of interest were examined using specific antibodies.

Collected cells were lysed using radio-immune precipitation assay (RIPA) buffer and protein was quantified using the Bradford assay (BioRad, Hercules, CA), with 40 μg of protein separated by 4–20% SDS-PAGE (BioRad). Proteins were electro-transferred onto Immobilon membrane (Millipore) and were probed with polyclonal rabbit anti-BACH2 (D3T3G; Cell signaling technology, Beverly, MA) anti-SENP3 (D20A10; Cell signaling technology) antibody, monoclonal rabbit anti-FOXP3 (D25D4; Cell signaling technology), and monoclonal mouse/human/rat anti-β-actin antibody (R&D systems, Minneapolis, MN). Applicable HRP-conjugated secondary antibodies were used with ECL chemi-luminescence to observe relevant bands using ChemiDoc XRS + image system (BioRad). The band intensity was quantified using Image J.

Western blot examined protein expression using antibodies against MYXV M-T7 and Serp-1 (Biogen, Cambridge, MA); host p-Akt (Thr308) and Akt (Cell Signaling Technology, MA, USA); and host collagen I and IV (abcam, Cambridge, UK). β-Actin was used as the control. Crude membranes were prepared in lysis buffer (Hepes [10 mM], pH 7.4; NaCl [38 mM]; PMSF [25 μg/mL]; leupeptin [1 μg/mL]; and aprotinin [1 μg/mL]) and centrifuged at 33000 rpm for 1 h, and the pellet was resuspended. Tumor tissues were collected after virus infection, washed with 1× PBS, and subsequently lysed with lysis buffer containing protease inhibitors. Proteins were quantified using the Bradford protein assay (Beyotime, CHN), separated by 10–12% SDS-PAGE, and transferred to PVDF membranes (Millipore, Billerica, MA, USA), which were blocked with 5% non-fat dry milk and incubated with primary antibodies. Proteins were visualized by the ChemiDoc™ XRS image system (Bio-Rad) using the appropriate secondary antibodies conjugated to horseradish peroxidase.

For Western Blot (WB) samples, 50 µg of protein was separated on precast 4–12% gel. At the end of electrophoresis, the proteins were stained with Ponceau Solution. Non-specific protein-binding sites on the nitrocellulose blots were blocked with PBS containing 7% non-fat milk (PBS–milk) for 1 h at RT. The primary antibodies were diluted in PBS at the following dilutions: 1:1000 for p75NTR C-terminal, 1:200 for ABCG2 (BXP-53: sc-58224), and 1:1000 for GAPDH antibodies. The incubation of the nitrocellulose blots with these antibodies was performed overnight (4 °C) at RT. The blots were then washed three times with PBS and incubated for 4 h at RT with the appropriate peroxidase-conjugated antibodies diluted 1:1000 in PBS–milk. Following PBS washes, the peroxidase activity of the nitrocellulose-bound secondary antibodies was detected with the Clarity West-ern ECL chemiluminescent reagents (BioRad Laboratories), imaged by Chemidoc XRS+ Image System (BioRad Laboratories), and bands were quantified by gel densitometry using the Fiji (ImageJ 1.53c) software (NIH, Bethesda, MD, USA). All the whole Western blot figures can be found in the Supplementary Materials.