Immobilon p polyvinylidene difluoride pvdf membrane

Cells (3 × 10⁵) were plated into six-well culture plates to achieve 70%–80% confluency, and were washed in PBS and suspended in 100 μL of RIPA buffer (Pierce, Dallas, TX, USA). Supernatant protein concentrations were determined using the BCA protein assay kit (Pierce). Supernatant samples containing 30 μg total protein were resolved by 10% or 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) depending on the molecular weights of the target proteins, and were transferred to immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) by electroblotting, and then probed with anti-E-cadherin (Cat# sc-21791, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-N-cadherin (Cat# sc-53488, Santa Cruz Biotechnology), anti-Vimentin (Cat# gtx100619, GeneTex, Irvine, CA, USA), anti-SNAIL1 (Cat# gtx125918, GeneTex), anti-Twist (Cat# gtx127310, GeneTex), anti-ZEB1 (Cat# gtx105278, GeneTex), anti-STC2 (Cat# sc-14352, Santa Cruz Biotechnology), anti-ANXA2 (Cat# sc-9061, Santa Cruz Biotechnology), anti-NRP1 (Cat# 3725, Cell Signaling Technology, Danvers, MA, USA), anti-SMAD2 (Cat#: 3103, Cell Signaling Technology), or anti-GAPDH (Cell Signaling Technology) antibodies. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Blots were developed using an ECL kit (Merck Millipore, Billerica, MA, USA).

Proteins from mice embryos were separated by 10% SDS–PAGE and transferred electrophoretically onto Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were probed with Sox7 polyclonal antibody (Proteintech, 23925-1-AP) and GAPDH antibody (Wuhan Servicebio Technology, GB11002) followed by either anti-rabbit (Jackson) or anti-mouse (Jackson) IgG secondary antibodies conjugated to horseradish peroxidase, then detected with a chemiluminescence system (BioRad).

Cell lysates obtained as described above were mixed with SDS electrophoresis sample buffer containing 90 mM DDT, boiled and subjected to electrophoresis in 4–12% gradient Novex NuPAGE SDS-PAGE Gels (Invitrogen). Resolved proteins were transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, Etobicoke, ON, Canada), and membranes were blocked in 5% skim milk and probed with various antibodies. Primary antibodies were monoclonal α-NS1 [42 (link)], monoclonal α-NP [44 (link)] (gift from Dr. Mingyi Li, National Microbiology Labs), monoclonal α-M1 (Thermo Fisher, MA1-80736), polyclonal α-M2 (Thermo Fisher, PA5-32233), monoclonal α-beta-actin (Cell Signalling, 3700S, Danvers, MA, USA), α-NUMA1 (Bethyl Laboratory, A301-510A, Montgomery, TX, USA), α-PRPF19 (Bethyl Laboratory, A300-101A) and/or α-UTP6 (Thermo Fisher, PA5-21716). Primary antibodies were detected with HRP-linked polyclonal α-mouse (Cell Signalling, 7076S), polyclonal α-rabbit (Cell Signalling, 7074S) or monoclonal VeriBlot™ (Abcam, Ab131366, Toronto, ON, Canada) secondary antibodies and HRP signals were detected using enhanced chemiluminescence (ECL) reagent (prepared in house). Images were taken with an Alpha Innotech Fluor Chem^® Q Imaging System and minimally processed by Adobe® Photoshop^® software (San Jose, CA, USA).

Cell lysates were prepared to detect the protein expression of cleaved PARP1. Equal amounts of protein (25 μg) were boiled with Laemmli sample buffer (Bio-Rad, USA) for 5 min and electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels. Separated proteins were then transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, USA) and blocked with 5% skim milk (Millipore) in Tris-buffered saline (TBS) containing 0.01% Tween-20 (TBST) for 30 min. The membranes were washed three times for 30 min in TBST and incubated with PARP1 (1:1,000 dilution, v/v, Cat#9542, Cell Signaling Technology, USA) and β-actin (1:2,000 dilution, v/v, Cat#sc-47778, Santa Cruz Biotechnology, USA) antibody in TBST buffer at 4°C overnight. The membranes were washed once every 10 min for 1 h in TBST and then incubated with secondary anti-rabbit (1:2,000 dilution, v/v, Cat#ADI-SAB-300, Enzo Life Sciences, USA) and anti-mouse (1:2,000 dilution, v/v, Cat#ADI-SAB-100, Enzo Life Sciences) HRP-conjugated antibodies for 1 h in TBST at room temperature. Primary and secondary antibodies were diluted with TBST and washed once every 10 min for 1 h. For visualization of the blots, the membranes were developed using D-Plus ECL Pico solution (Dongin Biotech, Korea).

All antibodies were purchased from Cell Signaling Technology (Danvers, MA). Total cell lysates were prepared, and immunoblotting was conducted as described previously [11 (link)]. Briefly, cells were cultured until subconfluent, rinsed with phosphate-buffered saline (PBS), lysed in sodium dodecyl sulfate (SDS) sample buffer, and homogenized. The total cell lysate (10 μg) was subjected to SDS polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). After blocking with 5% nonfat dry milk, membranes were incubated with primary antibodies, washed with PBS, and reacted with secondary antibodies (Cell Signaling Technology), and signals visualized using ECL reagent (Clarity, Bio-Rad, Hercules, CA) and film or detected by an ImageQuant Imager (GE Healthcare Bio-Sciences, Tokyo, Japan).