Western bolt experiments were performed with a primary antibody for TLR4 (Cell Signaling, 2219), Cleaved caspase-8 (Cell Signaling, 8592), RIP3 (ProScience, 2283), phospho RIP3 (Cell Signaling, 57220), MLKL (Santa Cruz Biotech, sc-165025), phospho MLKL (Cell Signaling, 37333), TNFR1 (Santa Cruz Biotech, sc8436) and βactin (cell signaling, 4970/58169) overnight at 4 °C coupled with appropriate secondary antibody as previously described3 (link). ECL was used as a method of detection. Chemiluminescence was recorded with an Image Station 440CF and results analyzed with the 1D Image Software (Kodak Digital Science, Rochester, NY).
Image station 440cf
Manufactured by Kodak
Sourced in United States, Sweden
The Image Station 440CF is a lab equipment product by Kodak. It is designed to capture, process, and output digital images. The product features a high-resolution scanning system and advanced image processing capabilities.
Automatically generated - may contain errors
Lab products found in correlation
1d image software, by Kodak (8 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Hrp immunstar detection kit, by Bio-Rad (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
1d image analysis software, by Kodak (3 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (3 mentions)
Supersignal west pico, by Thermo Fisher Scientific (3 mentions)
Rabbit anti nox4, by Novus Biologicals (2 mentions)
Iblot system, by Thermo Fisher Scientific (2 mentions)
Bis tris midi gel, by Thermo Fisher Scientific (2 mentions)
Mouse anti gapdh, by Merck Group (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Southern Biotech (2 mentions)
Laemmli buffer, by Merck Group (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Gapdh, by Abcam (2 mentions)
Glut5, by Merck Group (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Gelred nucleic acid gel stain, by Biotium (2 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (2 mentions)
45 protocols using image station 440cf
1
Western Blot Analysis of Necroptosis Markers
2
Whole Cell Extract Immunoblotting Protocol
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Whole cell extracts (WCE) were prepared from cells grown in YPD at 30 °C to approximately 3 × 107 cells/mL using trichloroacetic acid as previously described [54 (link)]. Equal amounts of WCE were separated by 12.5 or 15 % acrylamide SDS-PAGE, transferred to nitrocellulose (Whatman), and assayed by immunoblotting. The antibodies used to detect H3, H2B, Spt6, Spt16, Pob3, TAG, HA, Myc, and G6DPH were as follows: anti-H3 (1:30,000, described in [55 (link)] 1), anti-H2B (1:2500, Active Motif), anti-Spt6 (1:1000, gift from Tim Formosa), anti-Spt16 (1:500, gift from Tim Formosa), anti-Pob3 (1:2000, gift from Tim Formosa), anti-TAP (1:2000, Sigma), anti-HA (1:2000, Santa Cruz), anti-Myc (1:1000, Santa Cruz), and anti-GAPDH (1:50,000, Sigma). After incubation with HRP-conjugated IgG or secondary antibody (1:5000; GE Healthcare), the immunoreactive proteins were visualized by enhanced chemiluminescence detection (Perkin-Elmer) using a Kodak image station 440CF. Protein levels were calculated by measuring their signal intensities in these Western blots using Kodak ID 3.6 software and normalizing these values to those obtained for the G6PDH control.
3
Western Blot Protein Detection Protocol
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The protocols of the Western blot were performed as described by Hanson et al. [32 (link)]. Briefly, protein extracts, quantified by a Bradford Protein Assay (Bio-Rad Laboratories, CA, USA), underwent SDS-polyacrylamide gel electrophoresis and were transferred to Immobilon-P membranes. The following antibodies were used: rabbit anti-ERK1/2, goat anti-Matrin3, goat anti-βactin, anti-p22phox (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1 : 500, rabbit anti-Akt1, rabbit anti-Rac1, and rabbit anti-ERK1/2 (Cell Signalling Technology, Beverly, MA, USA), mouse anti-tubulin, rabbit anti-Nox1, and mouse anti-sc-35 (Sigma Aldrich St. Louis, MO, USA), rabbit anti-Nox4 (Novus Biologicals, CO, USA), rabbit anti-Nox2, and mouse anti-pH2A(Ser139) (Millipore, Billerica, MA, USA) diluted 1 : 1000; peroxidase-labelled anti-rabbit, mouse and goat secondary antibodies diluted 1 : 3000 (Pierce Antibodies, Thermo Scientific; Rockford, IL, USA). Ab dilution was performed in TBS-T pH 7.6 containing 3% BSA. The membranes were visualized using Supersignal substrate chemiluminescence detection kit (Pierce, Rockford, IL, USA). Anti-βactin antibody was used as control of protein loading. Quantization of the signal was obtained by chemiluminescence detection on a Kodak Image Station 440CF and analysis with the Kodak 1D Image software.
4
Hippocampal ALK1 Protein Expression
5
Western Blot Analysis of Hippocampal Proteins
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Forty µg of hippocampal protein per sample was subjected to PAGE electrophoresis using 4–12% Bis-Tris Midi Gel (Invitrogen) and transferred to a blotting membrane with the iBlot system (Invitrogen). The membrane probed with Goat anti-CHAT (1∶1000, Millipore) was blocked with Western Blocker Solution (Sigma). All other membranes were blocked with 5% milk in TBS/1.5% Tween (TBS-T), washed with TBS-T, and probed overnight with either rabbit anti-p75NTR (1∶3000, Advanced Targeting Systems), mouse anti-GFAP (1∶1000, Cell Signaling Technology), rabbit anti-TrkA (1∶1000, Millipore), rabbit anti-DCX (1∶1000, Cell Signaling Technology), rat anti-ALK-1 (1∶1000, R & D Systems), rabbit anti-BMP9 (1∶1000, Abcam), or mouse anti-β-actin (1∶5000, Sigma). Following incubation with the primary antibody, blots were incubated in species-specific anti-IgG-HRP: anti-Rabbit-HRP (1∶4000, Bio-Rad), anti-Goat/Sheep-HRP (1∶2000; Sigma), or anti-mouse-HRP (1∶2000, Bio-Rad).
Reactive bands were detected with SuperSignal West Femto chemiluminescent substrate (Pierce, Rockford IL). Chemiluminescence was captured with a Kodak ImageStation 440CF and the band intensities were quantified with Kodak 1D Image Analysis software.
Reactive bands were detected with SuperSignal West Femto chemiluminescent substrate (Pierce, Rockford IL). Chemiluminescence was captured with a Kodak ImageStation 440CF and the band intensities were quantified with Kodak 1D Image Analysis software.
6
Evaluating Intestinal Barrier Function
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Whole protein, including Occludin and TJP-1, which are responsible for maintaining the intestinal barrier function [72 (link)], and TLR4 and NF-κB signaling pathway, which are highly crucial for modulating inflammatory responses [52 (link)], from the liver and cecum mucous were lysed by RIPA lysis buffer (LifeTechnologies Inc., USA) supplemented with protease inhibitor cocktail (Roche, USA). The protein concentration in the tissue lysate was measured with BCA. Proteins were loaded onto the SDS-PAGE gel (BioRad) and electrophoresed and analyzed by WB using antibodies against TLR4 (BA1717, Boster, Wuhan, China), MYD88 (abs135682, Absin, Shanghai, China), IKBα (D120138, Sangon Biotech, Shanghai, China), p-IKBα (D151548, Sangon Biotech, Shanghai, China), p-p65 (HZ4902812, TW reagent, Shanghai, China), TJP-1 (mAb13663, Cell Signaling, USA), Occludin (ab167161, Abcam, USA), and actin (60008, Proteintech, USA). The bands were detected using the chemiluminescence kits (Amersham Biosciences, UK). Chemiluminescence was recorded with an Image Station 440CF, and results were analyzed with the 1DImage Software (Kodak Digital Science, Rochester, NY, USA).
7
Cell Lysis and Western Blot Protocol
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Cell extracts were obtained as described by Beretti et al. [21 (link)]. Briefly, subconfluent cells were extracted by the addition of AT lysis buffer (20 mM Tris-Cl, pH 7.0; 1% Nonidet P-40; 150 mM NaCl; 10% glycerol; 10 mM EDTA; 20 mM NaF; 5 mM sodium pyrophosphate; and 1 mM Na3VO4) and freshly added protease inhibitor cocktail at 4°C for 30 min. Lysates were sonicated, cleared by centrifugation, immediately boiled in SDS sample buffer, and centrifuged. Supernatants were loaded onto SDS-polyacrylamide gel, blotted on Immobilon-P membranes, and processed by western blot with the indicated antibodies, detected by SuperSignal substrate chemiluminescence detection kit. Quantitation of the signal was obtained by chemiluminescence detection on a Kodak Image Station 440CF and analysis with the Kodak 1D Image software. Primary antibodies were raised against the following molecules: rabbit-p16 and rabbit-β-actin.
8
Western Blot Analysis of Mouse Tissue Proteins
9
Western Blot Analysis of Muscle Signaling Proteins
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Protein lysates were prepared from mouse tissue or C2C12 cells using lysis buffer containing 0.3% Triton X-. Protein content was determined by the BCA protein assay (Pierce, Rockford, IL). Total protein (50 μg) was separated by SDS-PAGE [10% (w/v)] and transferred to PVDF membranes (Bio-Rad, Hercules, CA). Membranes were first blocked for 1 hat 25°C in 4% (w/v) instant milk dissolved in 0.1% Tris-buffered saline with Tween 20 TBS (TTBS) and incubated with the following primary rabbit-raised antibodies (1:1,000 dilution in TTBS): Myostatin (19142-1-AP, Proteintech; RRID:AB_10638615 ), AMPD1 (19780-1-AP; RRID:AB_10644281 ), GLUL (80,636, Cell Signaling; RRID:AB_2799956 ), pIRS1Ser636/639 (2388, Cell Signaling; RRID:AB_330339 ), IRS1 (2382, Cell Signaling; RRID:AB_330333 ), pAKTSer473 (4058 Cell Signaling; RRID:AB_331168 ), AKT (9272, Cell signaling; RRID:AB_32982 ) and GAPDH (5174, Cell Signaling; RRID:AB_10622025 ) and visualized using an anti-rabbit (no. 7074) horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000, Cell Signaling) using the HRP Immunstar detection kit (Bio-Rad). Chemiluminescence was recorded with an Image Station 440CF, and results were analyzed with the 1D Image software (Kodak Digital Science, Rochester, NY). Data for proteins of interest are expressed normalized to GAPDH expression.
10
Quantification of LRP1 Protein Levels
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Hippocampi from control and XR9576-treated mice were lysed in buffer containing 150 mM NaCl, 50 mM Tris, 0.5% deoxycholic acid, 1% Triton X-100, 0.1% SDS, 2.5 mM EDTA, and broad spectrum protease inhibitors. Protein concentration was determined in each sample using a Micro BCA Protein Assay kit (Thermo Fisher Scientific). 15 µg of protein for each sample was used for SDS-PAGE using 4–12% Bis-Tris gels with MES running buffer (Life Technologies). Rabbit polyclonal anti-LRP1 antibody was produced in G. Bu’s laboratory (Mayo Clinic, Jacksonville, FL), followed by goat anti–rabbit HRP (Santa Cruz Biotechnology). As a loading control, blots were stripped and reprobed with mouse anti-GAPDH (Sigma-Aldrich) and sheep anti–mouse peroxidase (GE Life Sciences). Blots were imaged on a ImageStation 440CF (Kodak) and bands quantified using 1D image analysis software (Kodak). LRP1 bands were normalized to GAPDH bands, and then for each gel the control-treated and XR9576-treated tissues were normalized to the mean of control bands intensity. LRP1 protein levels were compared between groups by unpaired Student’s t test.
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