Western blots were performed as previously described (Ramos et al. 2019 (link)). Briefly, cellular extracts and purified protein samples were fractionated on NuPAGE Bis-Tris polyacrylamide gels (Thermo Scientific) followed by transfer to Immobilon FL PVDF membrane (Millipore) for immunoblotting. For analysis of skin fibroblasts, 1 × 106 cells were harvested and proteins were extracted using radioisotope immunoprecipitation assay (RIPA) buffer (50 mM TrisHCl, pH 7.5, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA). Antibodies were against the following proteins : 6xHis tag (MA1-21315, Thermo Fisher), GFP (sc-9996, Santa Cruz Biotechnology), Strep-tag II-tag (NC9261069, Thermo Fisher), ADAT3 (Abcam, ab192987), ADAT3 (H00113179-B01P, Abnova), ADAT2 (ab135429, Abcam), and actin (CST). Primary antibodies were detected using IRDye 800CW Goat anti-Mouse IgG (SA5-35521, Thermofisher) or Rabbit (SA5-35571, Thermofisher) or Rat (925-32219, LI-COR Biosciences), or IRDye 680RD Goat anti-Mouse IgG (926-68070, LI-COR Biosciences) or Rabbit (925-68071). Immunoblots were scanned using direct infrared fluorescence via the Odyssey System (LI-COR Biosciences).
Sa5 35571
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SA5-35571 is a specialized laboratory instrument designed for general scientific applications. It features a compact and durable construction, making it suitable for use in various laboratory settings. The core function of this product is to provide consistent and reliable performance for the intended research or analytical tasks.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey system, by LI COR (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab96051, by Abcam (2 mentions)
A31573, by Merck Group (2 mentions)
A21206, by Thermo Fisher Scientific (2 mentions)
A21202, by Merck Group (2 mentions)
Sc 50509, by Proteintech (2 mentions)
A31572, by Merck Group (2 mentions)
A31570, by Merck Group (2 mentions)
Anti atrx, by Santa Cruz Biotechnology (2 mentions)
Ma1 21315, by Thermo Fisher Scientific (1 mentions)
Immobilon fl pvdf membrane, by Merck Group (1 mentions)
Gfp sc 9996, by Santa Cruz Biotechnology (1 mentions)
Nupage bis tris polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
6xhis tag, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Irdye 680rd goat anti mouse igg, by LI COR (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Image studio 3, by LI COR (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
8 protocols using sa5 35571
1
Western blot analysis of skin fibroblasts
2
Protein detection on PVDF membrane
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Samples were loaded onto SERVAGelTM TG PRiME 8–16% precast gels and transferred on polyvinylidene difluoride (PVDF) membranes (Bio-RAD). Proteins were detected with primary antibodies anti-St1SctP (InvJ)17 (1 : 2000) or M2 anti-FLAG (1 : 10,000) (Sigma-Aldrich, F3165). Secondary antibodies (ThermoFisher, SA5-35571) were goat anti-mouse IgG Dylight 800 conjugate (1 : 5000). Scanning of the PVDF membranes and image analysis was performed with a Li-Cor Odyssey system and Image Studio 3.1 (Li-Cor).
3
Quantitative Protein Analysis of Cellular Stress Pathways
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Cells were harvested 72h after treatment and centrifuged at 500g for 5 min. media and trypsin was eluted and cells were resuspended in 50–100ul of RIPA buffer. A Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA) was used according to manufacturer’s protocol to quantitate total protein in each sample. 20µg of protein sample was resolved using a SDS-page 15% acrylamide-bis gel and transferred to a nitrocellulose membrane (Advansta, San Jose, CA). Membrane was immunostained using rabbit polyclonal anti-survivin (NB500-201, NOVUS Biologicals, 1:1000) for detection of all survivin isoforms, mouse monoclonal anti-p53 (1:1000) (DO-1, sc126, Santa Cruz Biotech), mouse monoclonal anti-YY1 (1:1000) (H-10, sc-7341 Santa Cruz Biotech), rabbit polyclonal anti-sp1 (1:1000) (PEP-2 sc59, Santa Cruz Biotech) and loading controls rabbit monoclonal anti-β-actin (1:5000) (D6A8, Cell Signaling) and mouse monoclonal anti-GAPDH (1:1000) (O411, sc-47724, Santa Cruz Biotech). Dylight 800 Goat-anti-mouse IGG (1:20000) (SA535521, Invitrogen), and goat-anti-rabbit IGG antibodies (1:20000) (SA5-35571, Thermo Scientific) were used for secondary stain and quantitation via Odessey CL-x (LI-COR Lincoln, NE).
4
Lung Protein Extraction and Immunoblotting
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Total protein was extracted from the right lung tissues using RIPA buffer (Meilunbio, Dalian, China). Proteins were separated by 10% or 12% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, USA), which was blocked with 5% fat-free milk in TBST solution for 1 h at room temperature. The membranes were incubated with primary antibodies against LC3B (Abmart), Beclin1 (Abmart), p62 (Abmart), NF-κB p65 (Abmart), NLRP3 (Wanleibio), ASC (Cell Signaling Technology, Inc. MA, USA), pro-caspase1 (Abcam, Cambridge, UK), caspase1 p20 (Wanleibio), and mature-IL-1β (Wanleibio) at 4°C overnight. The membranes were washed with TBST and incubated with a secondary antibody (SA5-35571, Thermo Fisher, USA) at room temperature for 70 min. Immunoreactive images were detected using the Odyssey system (LI-COR Biosciences, USA) and analyzed using the ImageJ software.
5
Cerebral Cortex and Gastric ChAT Expression
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A Western blot analysis was performed to assess the expression of ChAT protein in the cerebral cortex on the infarcted side and in gastric tissues. The total protein concentration in each group was determined using the BCA Protein Assay Kit (G2026-200T, Servicebio). Subsequently, 10 μg of each sample was loaded onto a 10 % SDS-PAGE gel for electrophoresis. Following electrophoresis, the proteins were transferred onto PVDF membranes (IPVH00010, Millipore) and blocked with 5 % skim milk for 1 h at room temperature. The membranes were then incubated overnight at 4 °C on a shaker with primary antibodies, including ChAT (1:1000, ab181023, Abcam), α7nAchR (1:1000, ab216485, Abcam), and β-actin (1:1000, GB15003, Servicebio). On the following day, the membranes were incubated with a fluorescent secondary antibody (1:10000, SA5-35571, Invitrogen) and visualized using an Odyssey imager.
6
Antibody Validation for Immunofluorescence and Western Blotting
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The following antibodies were used for immunofluorescence or western blotting: Primary rabbit polyclonal: anti-actin (Sigma-Aldrich, A5060), anti-Daxx (Upstate, 07–471), anti-ATRX (Santa Cruz, H300), anti-PML (Bethyl Laboratories, A301-167A; Jena Biosciences, ABD-030), anti-Sp100 (GeneTex, GTX131569), anti-Mx1 (Santa Cruz, sc-50509; ProteinTech, 13750-1-AP), anti-ISG15 (ProteinTech, 15981-1-AP), anti-ISG54 (IFIT2, proteinTech, 12604-1-AP), and anti-histone H3 (abcam, ab1791). Primary mouse monoclonal: anti-HIRA (Millipore, 04–1488), anti-ICP0 (11060, [114 (link)]), anti-ICP4 (58s, [115 (link)]), anti-VP5 (DM165, [116 (link)]) anti-UL42 (Z1F11; [117 (link)]), and anti-PML (abcam, ab96051). Primary antibodies were detected using the following secondary antibodies: DyLight-680 or -800 conjugated goat anti-rabbit or -mouse (Thermo; 35568 and SA5-35571), Alexa -488, -555, or -647 conjugated donkey anti-rabbit or -mouse (Invitrogen; A21206, A21202, A31572, A31570, A31573, A31571), or HRP conjugated goat anti-mouse (Sigma-Aldrich, A4416).
7
Antibody panel for protein detection
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The following antibodies were used for immunofluorescence or western blotting: Primary rabbit polyclonal: anti-actin (Sigma-Aldrich, A5060), anti-Daxx (Upstate, 07–471), anti-ATRX (Santa Cruz, H300), anti-PML (Bethyl Laboratories, A301-167A; Jena Biosciences, ABD-030), anti-Sp100 (GeneTex, GTX131569), anti-SUMO2/3 (Abcam, ab22654), anti-Mx1 (Santa Cruz, sc-50509;ProteinTech, 13750-1-AP), anti-ISG15 (ProteinTech, 15981-1-AP), and anti-ISG54 (IFIT2, proteinTech, 12604-1-AP). Primary mouse monoclonal: anti-ICP0 (11060, [92 (link)]), anti-ICP4 (58s, [93 (link)]), anti-VP5 (DM165, [94 (link)]), anti-SUMO2/3 (Abcam, ab81371), anti-PML (abcam, ab96051), anti-IFI16 (abcam, ab55328; Santa Cruz, sc-8023). Primary antibodies were detected using the following secondary antibodies: DyLight-680 or -800 conjugated anti-rabbit or -mouse (Thermo; 35568 and SA5-35571), Alexa -488, -555, or -647 conjugated anti-rabbit, or -mouse (Invitrogen; A21206, A21202, A31572, A31570, A31573, A31571), HRP conjugated anti-mouse (Sigma-Aldrich, A4416).
8
Western Blot Detection Optimization
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Protein samples were run on 4–12% Bis-Tris gels (Life Technologies). After primary antibody incubation, blots were probed with 1:20,000 v/v dilution of either fluorescently labeled secondary antibodies (Life Technologies #A21058, Invitrogen #SA535571) in 2% bovine serum albumin in PBST or horseradish peroxidase-conjugated anti-rabbit secondary antibody (Veriblot Abcam #131366) in 5% non-fat milk in TBST for an hour at RT. Fluorescent images were developed using Odyssey. Veriblot-probed blots were treated with enhanced chemiluminescence substrate (Biorad #170–5060) for 5 min then developed on film. Original scans of all blots are included as a Source Data File.
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