Irdye 680rd goat anti rabbit

Ten µL aliquots of either a UC preparation or individual SEC fractions were analyzed by Western blot against human CD71. Membranes were probed with rabbit polyclonal anti-CD71 (ab84036) at 1:250 dilution for 1 hour at RT. A goat anti-rabbit IgG coupled to HRP (Sigma, A6154) was used at a dilution of 1:2,500 for 1 hour at RT. Revealing was performed using ECL Western Blotting Substrate (Pierce™) in ImageQuant LAS 4000 (GE Healthcare Life Sciences). Additionally, 20 µL aliquots of UC preparations were analyzed to confirm the presence of HSP70, GAPDH and stomatin in HuRex. Membranes were incubated for 1 hour at RT with primary antibodies anti-HSP70 (Santa Cruz Biotechnology, W27 sc-24) at 1:250 dilution, anti-GAPDH (Sigma, G9545) at 1:500 dilution or anti-stomatin (Invitrogen, PA5-30019) at 1:250 dilution. Subsequently, membranes were washed and incubated for 1 h at RT with the Li-Cor IRDye-labeled secondary antibodies IRDye® 800CW goat-anti-mouse (925-32210, Li-Cor Biosciences) at 1:15,000 dilution or IRDye® 680RD goat anti-rabbit (925-68021, Li-Cor Biosciences) at 1:20,000 dilution. Blots were analyzed with the Odyssey near-infrared system (Li-Cor Biosciences) having the intensity of 700 channel set up at 5 and the one of 800 channel at 7. Images were edited using the software Image J (NIH).

Cells were treated for 5 μM
compounds 11 and 85 or control (DMSO). The
treated cells were collected with a scraper at end of 48 h incubation
and lysates were prepared. Protein concentration was calculated with
a BCA assay. The protein levels were analyzed using protein electrophoresis
according to the manufacturer’s protocol for near-infrared
(NIR) Western Blot analysis (Mini-PROTEAN Tetra Cell Systems, Bio-Rad).
The samples (40 μg /well) were loaded onto TGX precast gels,
and protein transfer from gel to an LF-PVDF membrane was done using
Trans-Blot Turbo System (Bio-Rad). Primary antibodies (PARP, CST,
#9532S) and Calnexin (CST, #2679) and secondary antibodies IRDye 680RD
Goat Anti Rabbit (Li-Cor, # 92668071) and IRDye- 800CW Goat Anti Rabbit
(Licor, # 926e32211) were used for western blotting, and the proteins
were monitored in fluorescence system using Odyssey CLx- LICOR imaging
system.^{32 (link)}

Purified rVSV and rVSV-HTNV-GP were mixed with 4 × lithium dodecyl sulfate (LDS) sampling buffer (GenScript) and boiled at 100 °C for 10 min. Viral proteins were examined using SDS-PAGE followed by Coomassie Brilliant Blue G-250 (YEASEN) staining. For western blotting, viral proteins were separated and transferred onto a Poly (vinylidene fluoride) membrane, and incubated with mouse anti-Gn-1 (1:100), mouse anti-Gc-10 (1:1000), or rabbit anti-VSV-G (1:2000, abcam, 309106) antibody diluted in Tris-buffered saline containing 1% Tween-20 (TBST), followed by incubation with IRDye 680RD Goat anti-Mouse (1:10000, LI-COR, 926-68070) or IRDye 680RD Goat anti-Rabbit (1:10000, LI-COR, 926-32211), and imaged with an Odyssey Imager (LI-COR).

Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on Mini-Protean TGX Precast protein gels (Bio-Rad, Hercules, CA, USA) and transferred onto nitrocellulose membranes (Bio-Rad) using a Turbo transfer system (Bio-Rad). Membranes were blocked with 5% BSA T-BST (500 mM Tris HCl [pH 7.5], 1.5 M NaCl, 0.05% Tween 20). SpCas9-HF1 and β-actin were detected with antibodies 7A9-3A3 (Cell Signaling Technology, Danvers, MA, USA) and GTX629630-25 (GeneTex, Irvine, CA, USA), respectively. Membranes were probed with the fluorescent secondary antibodies IRDye 800CW goat anti-mouse (925-32210, LI-COR Biosciences, Lincoln, NE, USA) and IRDye 680RD goat anti-rabbit (925-68071, LI-COR Biosciences), respectively, and bands were visualized with a ChemiDoc MP Imaging System (Bio-Rad).

NSCLC cells were seeded in 100 mm dishes (300,000 cells/dish) overnight and treated with indicated treatments. Cell culture medium was changed one day after treatment. Three days post treatment, cells were lysed in RIPA buffer. Lysate was sonicated and centrifuged (21,130 x g for 15 min) and supernatant was collected. Amount of total protein was quantified using BCA. 30 μg of proteins (per sample) were mixed with 4X Novex NuPAGE LDS sample buffer and beta-mercaptoethanol (10% final concentration). Samples were denatured for 5 min at 95 ^oC and loaded onto gel (NuPAGE) for electrophoresis. Proteins were then transferred onto PVDF-FL membrane and blocked with LI-COR Intercept (TBS) blocking buffer. Membranes were incubated with primary antibodies overnight (Phospho-NF-kB p65 (Ser536) (93H1) antibody #3033, dilution 1:1000; Phospo-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody #9101, dilution 1:1000; β-Actin (8H10D10) antibody #3700, dilution 1:1000) at 4 ^oC. Next day, membranes were rinsed with TBS-T and IRDye conjugated secondary antibodies (IRDye 680RD Goat anti-Rabbit (LI-COR; 926-68071) and IRDye 800CW Goat anti-Mouse (LI-COR; 926-32210); or IRDye 800CW Donkey anti-Rabbit (LI-COR; 926-32213) and IRDye 680RD Goat anti-Mouse (LI-COR; 926-68070)) were added (dilutions 1:10,000) for 1 hour under rocking at room temperature. Membranes were scanned on a LI-COR Odyssey imaging system.