Protein extraction Kit (Beyotime, Shanghai, China) was used to extract total proteins. Then, 25μg proteins were separated by SDS-PAGE gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skimmed milk, the membranes were probed with primary antibodies against HSPA12B (1:1000, HPA015639, Sigma-Aldrich), PI3K (p85α) (1:1000, ab191606 Abcam, Cambridge, UK), p-PI3K (p85α, phospho Y607) (1:1000, ab182651, Abcam), AKT (1:1000, #4685, Cell signaling, Danvers, MA, USA), p-AKT (Ser473, 1:1000, #9271, Cell signaling), mTOR (1:1000, #2983, Cell signaling), p-mTOR (Ser2448, 1:1000, #5536, Cell signaling), and β-actin (1:1000, AM4302, ThermoFisher). The species-matched HRP-conjugated secondary antibodies were used (1:10,000). Protein bands were detected by the Odyssey imaging system (LI-COR, Lincoln, NE, USA).
Am4302
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The AM4302 is a lab equipment product from Thermo Fisher Scientific. It is a multipurpose instrument designed for various laboratory applications. The core function of the AM4302 is to perform precise measurements and analysis, but a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imaging system, by LI COR (2 mentions)
β actin, by Thermo Fisher Scientific (2 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (2 mentions)
Mabn52, by Merck Group (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Thermo Fisher Scientific (2 mentions)
Protein extraction kit, by Beyotime (1 mentions)
Ab182651, by Abcam (1 mentions)
Hpa015639, by Merck Group (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Recombinant rat ggt 2, by Jena Biosciences (1 mentions)
Fish his rep1, by Jena Biosciences (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ibright fl1000, by Thermo Fisher Scientific (1 mentions)
D2944, by Merck Group (1 mentions)
Modified bradford assay, by Bio-Rad (1 mentions)
Ab5594, by Abcam (1 mentions)
13 protocols using am4302
1
Western Blot Analysis of Signaling Proteins
2
Prenylation of RAB6A Protein
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The prenylation reactions were set up using 20 μg of total cell lysate, recombinant rat GGT-II (2 μM; Jena Biosciences, Jena, Germany), recombinant human RAB6A (4 μM; Jena Biosciences, Jena, Germany), and biotin-labeled geranyl pyrophosphate (B-GPP, 5 μM; Jena Biosciences, Jena, Germany) as lipid donor in prenylation buffer. All reactions were supplemented with fresh guanosine 5′-diphosphate (GDP; 20 mM; Merck Millipore, Watford, UK) and DTT (1 mM; Thermo Fisher Scientific, Loughborough, UK). Positive controls were prepared using untransduced cell lysate spiked with a recombinant REP1 protein (fish His-REP1; Jena Biosciences, Jena, Germany). The reactions were incubated for 2 h at 37°C and then stopped by addition of Laemmli sample buffer. Reaction products were subjected to SDS-PAGE and immunoblot analysis as per the protocol previously described in Patrício et al.15 (link) Membranes were incubated separately for detection of human REP1 (MABN52; 1:2,500; Merck Millipore, Watford, UK) and β-actin (AM4302; 1:50,000; Thermo Fisher Scientific, Loughborough, UK), which were detected using a horseradish peroxidase (HRP)-labeled secondary antibody (1:10,000). The incorporation of biotinylated lipid donor into the RAB6A substrate was detected by direct incubation with streptavidin-HRP (1:10,000; Thermo Fisher Scientific, Loughborough, UK).
3
Protein Isolation and Western Blot Analysis
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Total protein was isolated and quantified using a modified Bradford assay (Bio‐Rad). Western blot analyses were performed using the following primary antibodies: mouse anti‐COASY (Santa Cruz sc‐393812, 1/1,000), rat anti‐D1 dopamine receptor (Sigma D2944, 1/1,000), mouse anti‐TFR1 (ThermoFisher 13‐6800, 1/3,000), rabbit anti‐pyruvate dehydrogenase (Cell Signaling 3205, 1/3,000), rabbit anti‐VDAC (Cell Signaling 4661, 1/5,000), mouse anti‐MSH6 (BD 610918, 1/10,000), and mouse anti‐β actin (ThermoFisher AM4302, 1/10,000). Most of the primary antibody binding was visualized with the traditional method using a HRP‐conjugated secondary antibody (Jackson ImmunoResearch, 1/10,000) and ECL substrate (SuperSignal™ West Pico PLUS, ThermoFisher). However, anti‐DRD1 and its loading control (anti‐MSH6) were visualized using a biotinylated secondary antibody and Alexa Fluor™ 488‐conjugated streptavidin (ThermoFisher, 1/4,000). The fluorescent Western blot signal was captured using iBright™ FL1000 (ThermoFisher).
4
Quantifying Protein Levels in Stroke Tissue
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The protein levels of HSP27, pHSP27, and G6PD in infarcted cortical tissue from each group were determined by immunoblotting according to a previously described method [6 (link)]. Five percent bovine serum albumin (BSA, Nacalai Tesque) was used for blocking, and primary antibodies were diluted in Can Get Signal (TOYOBO) at 4 °C overnight. The following antibodies were used: rabbit polyclonal antibodies against G6PD (1:1000, #8866, Cell Signaling Technologies), HSP27 (1:1000, #2442, Cell Signaling Technologies), and pHSP27 (S85) (1:4000, ab5594, Abcam) and a mouse monoclonal antibody against β-actin (1:4000, AM4302, Thermo Fisher Scientific). The blots were quantified using densitometric analysis performed with NIH ImageJ software (ver. 1.51, https://imagej.nih.gov/ij/ ). β-actin was used as a loading control. The experiments were repeated two times.
5
Western Blot Analysis of REP1 Protein
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Reaction products were subjected to SDS-PAGE on 10% pre-cast polyacrylamide gel (Criterion, Bio-Rad, Hertfordshire, UK), transferred to a PVDF membrane (TransBlot Turbo, Bio-Rad, Hertfordshire, UK), and blocked with blocking buffer (PBS+0.1% Tween20 [PBST]+3% BSA) for 45 min. For protein expression, membranes were incubated separately for anti-β-actin (AM4302, Thermo Fisher Scientific, Loughborough, UK; 1:50,000) and anti-human REP1 (MABN52, Merck Millipore, Watford, UK; 1:2,500) primary antibodies in blocking buffer for 1 hr under agitation. Membranes were washed 3× for 7 min with PBST, incubated with horseradish peroxidase (HRP)-labeled secondary antibody for 30 min in blocking buffer (1:10,000), washed again as before, and detected using Clarity ECL (Bio-Rad, Hertfordshire, UK) and an Odyssey Imaging System (LI-COR Biosciences, Cambridge, UK). The incorporation of biotinylated lipid donor into the appropriate Rab substrate was detected by direct incubation with streptavidin-HRP (Thermo Fisher Scientific, Loughborough, UK) for 30 min. Densitometry data analysis was performed using ImageStudio Lite software (LI-COR Biosciences, Cambridge, UK).
6
Integrin and Myosin Light Chain Signaling
7
Western Blot Analysis of HBcAg, Cytoskeletal and Signaling Proteins
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Pre-cast 4–20% polyacrylamide gels (Mini-Protein TGX, Biorad, Hercules, CA, USA were used to separate proteins (Mini-Protein Tetra Cell, Biorad) and transferred onto nitrocellulose membranes using the Transblot-Turbo instrument (Biorad). The membranes were air-dried and blocked overnight in 1% skim milk at 4 °C. The following day, the membranes were incubated in primary antibody for 1 h and bound antibody detected with secondary antibody and ECL (Western Lightening Plus ECL, Perkin Elmer, Waltham, MA, USA). Primary antibody used were mouse anti-HBcAg [C1] (1:1000, Abcam ab8637, Cambridge, UK), mouse anti-αTubulin (1:1000, Abcam ab7291), mouse anti-active β-catenin (1:1000, Merck Millipore 05-665) and mouse anti-β-actin (1:5000, ThermoFisher AM4302, Waltham, MA, USA). Secondary antibody was rabbit anti-mouse polyclonal antibody HRP (1:10,000 Dako P0260, Glostrup, Denmark).
8
Western Blot Analysis of NEIL3 Protein
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Protein was extracted from fresh tissue, measured using the bicinchoninic acid method, and separated on a 12% sodium dodecyl sulfate polyacrylamide gel via electrophoresis (cat. #XP00100BOX; Thermo, USA). The protein was then transferred onto polyvinyl di uoride membranes (cat. #88518; Thermo), and the membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20. After the membranes were incubated with rabbit anti-NEIL3 (1:2000 dilution; cat. #PA5-51022; Thermo) or β-actin overnight at 4 °C (cat. #AM4302; Thermo) and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:10000 dilution; cat. #A32733; Thermo) for 1 h. The enhanced chemiluminescence technique was used to develop the signals.
9
Quantification of ACE2 Expression in Lung Tissue
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Approximate 50 mg lung tissue was homogenized by an Ultra‐Turrax T10 tissue homogenizer (IKA, Germany) for 20 s at full speed in 600ul RIPA (89 900, Thermo Scientific, Waltham, MA, USA) with Protease Inhibitor Cocktail (87 786, Thermo Scientific). The homogenate was incubated on ice for 30 mins and centrifuged at 18 400 g for 20 min. The supernatant was aliquoted and stored at −80 degree. Before loaded to the gel, the total protein concentration was quantified using a commercial bicinchoninic acid (BCA) kit (PC0021; Solarbio, China) and measured at 562 nm. A total amount of 30 ug protein was loaded to SDS/PAGE (10% separating gel, 5% stacking gel) and blotted onto the polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The blot was cut and incubated with an anti‐ACE2 antibody (ab15348, Abcam, 1 : 1000) or anti‐β‐actin antibody (AM4302, Invitrogen, Waltham, MA, USA, 1 : 5000) overnight. After washing with phosphate‐buffered saline with Tween 20 (PBST), the blots were subjected to an HRP‐conjugated goat anti‐rabbit antibody (SA00001‐2; Proteintech, Rosemont, IL, USA, diluted in 1 : 5000) for 30 mins and visualized with ECL substrates (32106, Pierce) on BioDocAnalyze (BDA) digital (Analytik Jena, Germany). The bands were quantified with imagej (NIH, USA) as others had described [16 (link)].
10
Western Blot Analysis of Antioxidant Proteins
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Cells were lysed in ice-cold RIPA buffer with protease inhibitors (Thermo Fisher Scientific, PIA32955) followed by sonication in a water bath sonicator (Diagenode). Protein was quantified using the DC protein assay (Bio-Rad). Lysates were prepared with 6X SDS sample buffer containing 12% (v/v) β-ME (VWR) and separated on Bolt or NuPAGE 4%–12% Bis-Tris gels (Invitrogen). SDS-PAGE separation was followed by transfer to 0.45-μm nitrocellulose membranes (GE Healthcare). The membranes were blocked in 5% non-fat milk in TBS with 0.1% Tween 20 (TBST). For immunoblotting, the following antibodies diluted in 5% milk in TBST were used: KEAP1 (Millipore Sigma, RRID:AB_2921362, 1:2,000), NRF2 (Cell Signaling Technology, D1Z9C, RRID:AB_2715528, 1:1,000), NQO1 (Sigma-Aldrich, RRID:AB_1079501, 1:1,000), GCLC (Sana Cruz Biotechnology, H-5, RRID:AB_2736837, 1:1,000), xCT (Abcam, RRID:AB_778944, 1:1,000), GSR (Santa Cruz Biotechnology, RRID:AB_2295121, 1:1,000), β-actin (Invitrogen AM4302, RRID:AB_2536382, 1:100,000). HRP secondary antibodies used include goat anti-rabbit IgG (Jackson ImmunoResearch Labs, RRID: AB_2313567), goat anti-mouse IgG (Jackson ImmunoResearch Labs, RRID:AB_10015289), and goat anti-rat IgG (Jackson ImmunoResearch Labs, RRID:AB_2338128). Membranes were developed with Clarity ECL substrate (Bio-Rad) or a luminol-based homemade ECL substrate.
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