Alexa fluor 488 goat anti rabbit igg

After 24 h seeding on the sterile slides in 24-well plates, cells were washed with Phosphate Buffered Saline (PBS) for 3 times and fixed with 4% paraformaldehyde for 20 min. Cells on the slides were permeabilized with 0.2% Triton X-100 for 5 min and blocked with 5% BSA for 1 h. Then slides were incubated with the primary antibody (1:400) overnight at 4 °C. Followed by incubation with fluorescent secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG, 1:200, Cell Signaling Technology; Alexa Fluor 594 goat anti-rabbit IgG, 1:200, Cell Signaling Technology) for 1 h at room temperature without lighting, cell nuclei were stained with DAPI for 5 min. Slides were imaged by confocal microscopy (IX83, FLUOVIEW FV1200, Olympus).

Primary antibodies: Mouse anti-Flag (Cell Signaling 8146), rabbit anti-V5 (Cell Signaling 13202), rabbit anti-His (Cell Signaling 12698), anti-Sf-SR-C-N polyclonal antibodies were generated by immunizing rabbits with purified GST-SR-C-N. Secondary antibodies: goat anti-mouse IgG-HRP conjugate (Santa Cruz sc-2005), goat anti-rabbit IgG-HRP conjugate (Cell Signaling 7074), rabbit anti-GST (Polyclonal, Bioss bs-2735R). The primary antibodies and secondary antibodies were used at 1: 1000 for western blotting. For immunostaining assays, Anti-V5-Dylight 488 conjugate (Invitrogen MA5-15253-D488, 1:200) and Alexa Fluor 488 goat anti-rabbit IgG (Cell Signaling 4412, 1:200) were used.
Inhibitors: Dynasore (dynamin inhibitor, TargetMol T1848, 7.5μM), chlorpromazine (clathrin-mediated endocytosis inhibitor, Millipore 215921, 40μM), monodansylcadaverine (clathrin-mediated endocytosis inhibitor, Sigma D4008, 150μM), nystatin (sequesters cholesterol, Millipore 475914, 20μM), cholesterol-oxidase (oxidize cholesterol, Millipore 228230, 4unit/ml), amiloride (macropinocytosis inhibitor, Millipore 129876, 150μM), Cytochalasin D (macropinocytosis inhibitor, Millipore 250225, 500nM), LY294002 (broad PI(3)K inhibitor, Cell Signaling 9901s, 50μM), and wortmannin (broad PI(3)K inhibitor, Cell Signaling 9951s, 2μM). Cells were treated with the inhibitors for 1 h at 27°C before Vip3Aa-RFP was added.

Research was approved by the Columbia University Medical Center Institutional Review Board (IRB-AAAF2671) and informed written consent was obtained from all participants. Peripheral whole blood samples from healthy human volunteers were collected in heparin tube and irradiated with γ rays (0 and 4 Gy) using a Gammacell 40 ¹³⁷Cs irradiator (Atomic Energy of Canada, Ltd.). Peripheral blood was incubated in RPMI 1640 (Gibco) with 15% FBS and 2% Penicillin and Streptomycin at 37 °C. PBMCs were isolated from cultured blood samples (n = 3 ~ 4) 3 days after radiation exposure and were prepared for immunofluorescent labeling using the same protocol described above in the humanized mice model. Cells were incubated with the following antibodies: rabbit anti-FDXR, rabbit anti-DDB2 (Thermo Fisher Scientific), rabbit anti-ACTN1 (Cell Signaling Technology, Danvers, MA), and Alexa fluor 488 goat anti-rabbit IgG. Fluorescence levels of candidate proteins and percentages of positive cells based on maximum pixel intensity were measured and fold changes compared to baseline level were calculated. Quantification of radiation-induced protein expression levels was determined in non-apoptotic cells only by the image analysis software program^{48 (link)}, such that advanced apoptotic cells observed as a gross change in area and morphology of the DAPI-labeled nuclei, were not included for analysis.

Cells (1 × 10⁴/well) were cultured on 96-well culture plate (Corning, USA). After incubation with different test substances, the cells were fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X-100 for 15 min. Next, the cells were blocked with 5% BSA for 1 h and incubated with primary antibody overnight at 4 °C. Alexa-conjugated secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-rabbit IgG, Cell Signalling Technology, MA, USA) were applied and incubated at room temperature for 1 h. Cell nuclei were stained with DAPI (Cell Signalling Technology, MA, USA) for 10 min. Finally, the cells images were analysed by ImageXpress® Micro Confocal (Molecular Devices, USA).

Proteins were transfected into yeast cells using the Xfect™ Protein Transfection Reagent (Takara Bio Inc., Shiga, Japan), according to the Manufacturer’s instruction with modifications. Briefly, yeast cells (4.0 × 10⁷ cells/ml) suspended in PBS (pH 7.4) or 10 mM MES (pH 6.0) were mixed with the Xfect reaction buffer along with proteins, preincubated at room temperature for 30 min, and then washed with PBS to remove the reaction reagent. Proteins used were 4 µg β-Gal (accessory in Xfect reagent), 46 µg recombinant GFP (Abcam, Cambridge, CB2 0AX, UK), 5 µg Alexa Fluor 488 goat anti-rabbit IgG (Cell Signaling Technology, Danvers, Massachusetts, USA), and 50–70 µg recombinant NLS-TaqI (prepared in this study; see below). In experiments shown in Fig. 1b, protein/Xfect complexes absorbed on the extracellular membrane or cell wall were digested by 1% trypsin/PBS treatment at 37 °C for 5 min^{32 (link),40 (link)}.

Podocytes were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. After rinsing twice with PBS, cells were blocked with 2% bovine serum albumen (BSA) in PBS for 1 h at room temperature. Cells on coverglass were incubated with KCa1.1 (1184–1200) antibody (APC-107, Alomone Labs) at a 1:100 dilution in PBS with 2% BSA at 4°C overnight. Cells were rinsed three times with PBS containing 0.02% Tween 20. Secondary antibodies were diluted in PBS with 2% BSA and cells were incubated at room temperature for 1 h. Alexa Fluor 488 goat anti-rabbit IgG (1:500; Cell Signaling Technology, United States) served as the secondary antibody. After rinsing three times with PBS containing 0.02% Tween 20, cells were incubated with DAPI (2 μg/ml) for 1 min. Cells were rinsed three times with PBS containing 0.02% Tween 20 and cells on coverglass were mounted on microscope slides with Prolong Gold antifade reagent (Invitrogen, United States). Images were taken using a Carl Zeiss LSM710 confocal microscope and processed using Photoshop software (Adobe Systems, Inc., San Jose, CA, United States). All of the images were quantified with ImageJ software and normalized to the respective control.