Cells were lysed in RIPA buffer containing 1% NP-40, 150 mM NaCl, 50 mM tris-Cl (pH 8.0), and 1% SDS supplemented with cOmplete, EDTA-free protease inhibitor (11873580001, Roche). Protein concentration was determined by bicinchoninic acid assay (BCA) protein assay (#23227, Life Technologies), after which equal amounts of proteins were loaded and separated by polyacrylamide gel electrophoresis. The following antibodies were used: p53 (OP43, Millipore), p21 (ab109520, abcam), and Gapdh (10R-G109a, Fitzgerald).
10r g109a
Manufactured by Biosynth
Sourced in United Kingdom, United States
The 10R-G109A is a laboratory equipment designed for general purpose use. It is a versatile instrument that can be utilized in various scientific applications. The core function of this product is to provide a reliable and consistent platform for various experimental procedures. Further details on the intended use of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Mini protean tgx gel, by Bio-Rad (2 mentions)
Ab109520, by Abcam (1 mentions)
Rabbit anti 4e bp1, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Rabbit anti phospho 4ebp1, by Cell Signaling Technology (1 mentions)
Rabbit anti p70s6k, by Cell Signaling Technology (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Turbo transfer system, by Bio-Rad (1 mentions)
Goat anti rabbit irdye 800, by LI COR (1 mentions)
Donkey anti mouse irdye 680, by LI COR (1 mentions)
Mab2263, by Merck Group (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ab1670, by Abcam (1 mentions)
Ab129058, by Abcam (1 mentions)
Ap17961pu n, by OriGene (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by AAT Bioquest (1 mentions)
18 protocols using 10r g109a
1
Protein Extraction and Western Blot Analysis
2
Protein Extraction and Western Blot Analysis
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Cells were lysed in TNE lysis buffer consisting of 150 mmol/L NaCl, 0.5% Nonidet P‐40, 60 mmol/L Tris and 1 mmol/L EDTA, supplemented with 1× protease inhibitor cocktail (Nacalai Tesque) and PhosSTOP phosphatase inhibitor cocktail (Roche). Western blot analysis was performed as described previously.23 Primary antibodies used in this study were as follows: mouse anti–ALDH1A3 (0.5 μg/mL, GT926; GeneTex), rabbit anti–phospho‐p70S6 kinase (p70S6K, phosphorylated at T389) (1:1000, #9234; Cell Signaling Technology), rabbit anti–p70S6K (1:1000, #9202; Cell Signaling Technology), rabbit anti–phospho‐4E‐BP1 (phosphorylated at S65) (1:1000, #2855; Cell Signaling Technology), rabbit anti–4E‐BP1 (1:1000, #9644; Cell Signaling Technology) and mouse anti–GAPDH (0.02 μg/mL, 10R‐G109a; Fitzgerald).
3
Quantification of Hippocampal HP1BP3 Protein
4
Western Blot Analysis of Glutamate Receptors
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Surface protein fractions (60 µg) and their corresponding total protein samples (8 µg) were resolved in 10% SDS polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were blocked (50% Odyssey blocking buffer in PBS; Li-Cor, Lincoln, NE) for one hour at room temperature, followed by overnight incubation with the primary antibodies at 4°C. The primary antibodies consisted of mouse anti-glutamate receptor 1 N-terminus (1:1000, #MAB2263; Merck Millipore, Burlington, MA), rabbit anti-GluA2 (1:1.000, #MAB5306S; Cell Signaling Technology, Danvers, MA) and mouse anti-GAPDH (1:1,000,000, #10R-G109A; Fitzgerald Industries, Acton, MA) as loading control. Membranes were subsequently incubated with secondary antibodies for one hour at room temperature: goat anti-rabbit IRDye 800 (1:10,000; Li-Cor) and donkey anti-mouse IRDye 680 (1:10,000; Li-Cor). Membranes were visualised using the Odyssey Infrared Imaging System (Li-Cor), and protein bands were quantified using ImageJ (National Institutes of Health, Bethesda, MD). Raw intensity measures were normalised to GAPDH to control for loading differences.
5
Quantifying CXCR4, CXCR7, and 5T4 Expression
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Western blot (WB) analysis and immunofluorescence (IF) staining of cultured cells were performed as previously described [6, 7] using the following primary antibodies: goat anti-CXCR4 (ab1670, Abcam, WB, 1:1000; IF, 1:100); rabbit anti-CXCR7 (AP17961PU-N, Acris, WB, 1:300; IF, 1:100; ab138509, Abcam, WB, 1:1000); rabbit anti-5T4 (ab129058, Abcam, WB, 1:1000). Protein loading was controlled by additionally staining blots with anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (10R-G109A, Fitzgerald, 1:5000). Cell nuclei in immunostained cell cultures were visualized with 4′,6diamidino-2-phenylindole (DAPI; AAT Bioquest, Sunnyvale, CA) and inspected on a Zeiss confocal laser scan microscope (LSM).
6
Western Blot Analysis of Cardiomyocyte Proteins
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HL-1 cardiomyocytes or human tissue samples were lysed in radioimmunoprecipitation assay (RIPA) buffer containing PBS, Igecal ca-630, eoxycholic acid, and sodium dodecyl sulfate (SDS)2 (link),8 (link). In short, equal amounts of protein homogenates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membranes, and probed with antibodies directed against poly (ADP-Ribose) (PAR, 1:1000, BD bioscience, 551813), PARP1 (1:500, Santa Cruz, sc-25780), γH2AX (1:1000, Millipore, 05–636), Cav1.2 (1:200, Alomone Labs, ACC-003), Kv11.1 (1:400, Alomone Labs, APC-062), Kir3.1 (1:200, Alomone Labs, APC-005), β-actin (1:1000, Santa Cruz, sc-47778), or GAPDH (1:5000, Fitzgerald, 10R-G109A). Membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Dako). Signals were detected by the ECL detection method (Amersham) and quantified by densitometry (Syngene, Genetools). Original uncropped blots are available at the Supplementary Information section (Supplementary Figure 12 ).
7
Immunoblotting Antibody Validation
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The antibodies used for immunodetection by western blot were: PALB2 antibody rabbit pAb (A301-246A, Bethyl), monoclonal ANTI-FLAG® M2 antibody produced in mouse (F3165, Sigma), GAPDH antibody mouse mAb (10R-G109a, Fitzgerald), anti-Histone H3 antibody—Nuclear Loading Control and ChIP Grade (ab1791, Abcam), purified mouse anti-human Exportin-1/CRM1 (611832, BD Biosciences), mouse anti-GFP (11814460001, Roche).
8
Western Blotting Analysis of Protein Expression
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Protein extractions were performed and protein concentrations were determined as described previously [31 (link)]. Forty μg of total protein was loaded per lane on 4-20% Mini PROTEAN TGX Gels (Bio-Rad Laboratories, Hercules, CA, USA). Western blots were performed using anti-NUSAP1 (1:2,500; 12024-1-AP; Proteintech Group, Rosemont, IL, USA), anti-FAM101B (1:500; ab150350; Abcam, Cambridge, UK), or anti-GAPDH (1:4,000; 10R-G109A; Fitzgerald Industries International, Acton, MA, USA) primary antibodies; and Peroxidase AffiniPure Goat Anti-Mouse IgG, Light Chain Specific (1:10,000; 115-035-174 Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or Peroxidase IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, Light Chain Specific (1:5,000 or 1:10,000; 211-032-171; Jackson ImmunoResearch Laboratories, Inc.) secondary antibodies. Western blots were developed and stripped as described previously [31 (link)], and visualized using the Mini-Medical/90 film processor (AFP ImageWorks, Elmsford, NY, USA).
9
Protein Extraction and Detection in Cardiac Cells
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Whole-cell lysates of mouse LV and NRVMs were prepared in T-PER (Thermo) containing protease and phosphatase inhibitors (Roche). Nuclear fractions of heart 4 days following sham- or TAC procedures were prepared using NE-PER nuclear cytoplasmic extraction reagents according to the manufacturer’s instructions (Thermo). Equal amounts of protein (3–10 μg) were separated on a 4–20% SDS-PAGE gel (Bio-Rad), transferred to a nitrocellulose membrane, and immunoblotted to detect and quantify specific protein bands using an Odyssey scanner (LI-COR version 3)74 (link). Proteins were detected with a 1000-fold dilution of the following primary antibodies: FoxO1 (#2880, Cell Signaling and ab39670; Abcam); Phospho-FoxO1 (#9464, Cell Signaling), ERK (#4695, Cell Signaling); Phospho-ERK (#4370, Cell Signaling); 10,000-fold dilution of GAPDH (10R-G109a, Fitzgerald) and tubulin (ab6046; Abcam) and 500-dilution of Dio2 antibody (ab77481, Abcam) antibodies, respectively.
10
Western Blot Analysis of Dok-7 Protein
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Protein extracts from transfected HEK cells were separated by SDS-PAGE, transferred into nitrocellulose membranes, and incubated with rabbit antieDok-7 (H-77; dilution 1:400; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse antieglyceraldehyde-3-phosphate dehydrogenase (10R-G109a; dilution 1:3,000,000; Fitzgerald, Acton, MA;) antibodies. Secondary antibodies were donkey anti-mouse IgG IRDye 680 and goat anti-rabbit IgG IRDye 800 (926-32222 and 926-32211, respectively; LI-COR Biosciences, Lincoln, NE), diluted 1:10,000. Stained membranes were imaged with the Odyssey Imaging System (LI-COR Biosciences), and bands were quantified with the ImageJ software version 1.48a (NIH), normalizing for glyceraldehyde-3-phosphate dehydrogenase expression.
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