Ibind flex western device

Cells were collected and lysed with RIPA lysis buffer, and lysates were centrifuged at 10,000 × g for 20 min. Protein concentration of collected supernatants was measured by Quick Start Bradford Protein Assay (Bio-Rad). Equal 10 μg proteins were loaded into 10% TGX polyacrylamide gels (Bio-Rad). The proteins were electrophoresed at 200 V for 40 min and transferred to Poly Vinylidene Di-Fluoride membranes. Immunoblots were detected using iBind Flex Western Device (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. Anti-HNF1A rabbit polyclonal antibody (ab204306; Abcam) at a dilution of 1:150 and anti-Histone H3 rabbit monoclonal antibody (#4499, Cell Signaling Technology) at a dilution of 1:2000 were used as primary antibodies. The iBind Western System was run for 3 h with HRP-linked secondary antibody (#7074, Cell Signaling Technology) at a dilution of 1:2000. Protein expressions were visualized by Luminescent Image Analyzer LAS-3000 (Fujifilm Corporation, Tokyo, Japan). All experiments were independently performed in triplicate and a representative figure was shown.

siRNA (Santa Cruz Biotechnology) targeting cd44 was used to knock down its gene expression in the HCMEC/D3 cells. Cells were plated in six well plates and grown until confluent. A solution of 8 µL siRNA duplex, 8 µL siRNA transfection reagent, and 1 mL of siRNA transfection medium was introduced to the cells in each well. The cells were incubated for 5 hours at 37 °C after which 1 mL of EGM containing 2% penicillin/streptomycin and 10% fetal bovine serum was added without removing the transfection mixture. The cells were incubated for an additional 18 hours after which they were used for experiments. To verify knockdown, cellular protein was isolated using digestion in sample buffer with reducing agent, and separated using SDS-PAGE. Following transferm PVDF blots were incubated with an anti-cd44 primary (1:50) and horseradish peroxidase-conjugated secondary (1:4000) within an iBind Flex Western Device (Thermo Scientific). Blots were imaged in a chemiluminescent imager. To provide a loading control, gels were incubated in Coomassie Blue for 30 minutes following the transfer and representative bands were used to normalize blot signals.

HEK293T cells (2 × 10⁵) were seeded in 12-well plates (Techno Plastic Products). The next
day, at a confluence of 50–70%, the cells were transiently
transfected with a mixture of DNA and PEI (8 μL PEI/1000 ng
DNA). Forty-eight hours post-transfection, the cells were washed with
1 mL PBS and lysed in 100 μL 1 × Passive lysis buffer (Promega).
The cells were lysed for 20 min on ice and centrifuged for 15 min
at 14 000 rpm to remove cell debris. The total protein concentration
in the supernatant was determined using the BCA assay.
Proteins
from the supernatant were separated on 12% SDS-PAGE gels (200 V, 45
min) and transferred to a nitrocellulose membrane (350 mA, 60 min).
Membrane blocking, antibody binding, and membrane washing were performed
using an iBind Flex Western device (ThermoFisher) according to the
manufacturer’s protocol. The primary antibodies were mouse-anti
Myc (Cell Signaling Technology 2276; diluted 1:2000). The secondary
antibodies were HRP-conjugated goat antimouse IgG, diluted 1:3000
(Jackson ImmunoResearch 115-035-003). The secondary antibodies were
detected with an ECL Western blotting detection reagent (Super Signal
West Femto; ThermoFisher) according to the manufacturer’s protocol.

Cells were infected with CF33-GFP virus at MOI 5 in a 6-well plate. Medium from wells were collected at 24, 48, and 72 h and concentrated 10 folds using centrifugal filters (UFC801024; Amicon). Next, 25 μL of each sample was separated on a Criterion Precast polyacrylamide gel (3450028; BIO-RAD) then transferred to a PVDF membrane. The membrane was stained with anti-HMGB1 antibody (ab18256; Abcam) and donkey anti-rabbit antibody (926–32213; Li-COR) in iBind Flex FD solutions (1930520; Invitrogen) using iBind Flex Western Device (ThermoFisher) for 3 h. The membrane was then scanned using Azure C600 scanner (Azure Biosystems).

Total protein was extracted from cultured cells using RIPA buffer containing protease inhibitors (Roche, Indianapolis, IN, USA). The lysates were sonicated on ice and centrifuged at 14,000 × g for 15 min at 4 °C. The supernatant was collected, and protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). After mixing with NuPAGE LDS Sample Buffer (Thermo Fisher Scientific), cell lysates were denatured at 70 °C for 10 min, electrophoresed using NuPAGE Novex Tris-Acetate Gel 3–8% (Invitrogen) at 150 V for 75 min, and then transferred to PVDF membranes. The membranes were incubated with primary antibodies, which was followed by incubation with secondary antibody using the iBind Flex Western Device (Thermo Fisher Scientific). The following primary antibodies were used: rabbit anti-dystrophin (1:500, Abcam, Cambridge, UK; ab15277), mouse anti-myosin heavy chain (1:200, R&D, Minneapolis, USA; MAB4470), and mouse anti-α-tubulin (1:1000, Sigma; T6199). Histofine Simple Stain MAX-PO (1:100, NICHIREI BIOSCIENCE INC., Tokyo, Japan; 424151) was used as a secondary antibody. Proteins were detected using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, UK; RPN2232) and a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). Data were analysed using Image Lab 6.0 (Bio-Rad).